Abstract The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) 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. 47 fragments with diverse scaffolds were identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we performed deep mutational scanning of NS2B-NS3 protease. Merging fragment hits yields an extensive set of ‘mergers’, defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points that maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.

ABSTRACT Congenital Zika syndrome (CZS), the set of fetal and neonatal complications associated with Zika virus (ZIKV) infection in pregnancy, was first noted during the outbreak in the Americas in 2015-16. However, there was an unequal distribution of ZIKV cases and severe outcomes in all areas where ZIKV emerged in the Americas, demonstrating that the risk of CZS varied over space and time. Recently, we demonstrated that phenotypic heterogeneity existed between closely-related ZIKV strains. All ZIKV strains tested infected the placenta but varied in their capacity to cause overt fetal harm. Here, we further characterized the relative contributions of virus genotype and infecting dose of two phenotypically distinct ZIKV strains across multiple timepoints in gestation in pregnant mice that lack type-I interferon receptor function ( Ifnar1 -/- ). To better understand the underlying causes of adverse fetal outcomes, we used RNA sequencing to compare ZIKV-infected and uninfected tissues. We found that ZIKV infection triggers retinoic acid-inducible gene I (RIG-I)-like receptor-mediated activation of the interferon response at the maternal-fetal interface. However, modest chemical inhibition of RIG-I activation in the decidua and placenta did not protect against fetal demise. Instead, the fetal interferon response was significantly associated with fetal demise. Together, these findings suggest that the response to ZIKV at the maternal-fetal interface can vary depending on the infecting ZIKV genotype and dose, and that the fetal immune response is an important mediator of fetal harm. IMPORTANCE Previously, we used a mouse model of ZIKV infection during pregnancy to assess the pathogenic potential to the fetus of a panel of five, low-passage ZIKV strains representing the viral genetic diversity in the Americas. We found that phenotypic heterogeneity existed between these closely-related ZIKV strains. Here, we show that this heterogeneity is driven by retinoic acid-inducible gene I (RIG-I)-like receptor-mediated activation of the interferon response at the maternal-fetal interface. We used chemical inhibition of the RIG-I pathway and measured the transcriptional activity of interferon stimulated genes in fetuses to demonstrate that the fetal immune response may contribute to fetal demise.

The morphogenesis of enveloped viruses relies on the trafficking of transmembrane proteins through the secretory pathway to sites of virus envelopment. The first step in this pathway, their translocation into the endoplasmic reticulum, is therefore an attractive target for broad-spectrum intervention. Here, we tested if blockade of the Sec61 translocon by the Mycobacterium ulcerans exotoxin mycolactone, a potent inhibitor of Sec61, could block the production of virus glycoproteins and subsequent production of infectious virus from a range of human enveloped viruses: the DNA virus herpes simplex virus 1 (HSV1), and the RNA viruses, respiratory syncytial virus (RSV), influenza A virus (IAV), SARS coronavirus 2 (SARS CoV2) and Zika virus (ZIKV). In line with known translocation mechanisms, mycolactone blocked in vitro translocation and ectopic expression of type I transmembrane proteins but not type III, multipass or cytosolic proteins. Translocation of the type II protein RSV G was also blocked and although ectopically expressed G protein was detected, it was not glycosylated. Pretreatment of cells with mycolactone also blocked the synthesis of type I transmembrane proteins in infected cells and either the synthesis or glycosylation of type II transmembrane proteins, and the production of progeny from all viruses tested, while having no effect on virus entry or downstream synthesis of cytosolic proteins. While mycolactone treatment of HSV1 infected cells at various times after infection resulted in the immediate inhibition of virus production at the point of addition, IAV, RSV and ZIKV became resistant to the action of mycolactone surprisingly early in infection, and before virus glycoprotein synthesis was even detectable or virus production had begun. We therefore conclude that although inhibition of the translocation of virus transmembrane proteins through the Sec61 translocon can in principle block virus production, the morphogenesis of many enveloped RNA viruses requires only limited amounts of envelope proteins for successful propagation, providing novel insight into the biology of these viruses. Author Summary Many circulating human pathogens are enveloped viruses that all use the cellular secretory pathway to target their envelope proteins to cellular sites of virus particle assembly. The potential to target this pathway could therefore offer a novel broad-spectrum therapy for existing, emerging and as yet unknown human pathogens. Here we have targeted the initial step in this pathway using a highly potent Sec61 inhibitor, mycolactone, to carry out the first comprehensive assessment of translocation disruption on a range of enveloped human viruses from different virus families, including herpes simplex virus, influenza A virus and SARS-CoV2. Our results have shown that Sec61 inhibition blocks the onward trafficking of many virus envelope proteins that are essential to produce infectious virus at assembly sites. However, unexpectedly, we found that several of the viruses were resistant to the effects of this toxin when it was added early in infection, indicating that the synthesis of these essential virus proteins occurs earlier in infection than previously recognised. Hence, while this approach may not be suitable as a broad intervention strategy, it has revealed new information on virus biology and provides us with a novel tool for exploring a wide range of enveloped viruses.

The mosquito microbiota represents an intricate assemblage of microorganisms, comprising bacteria, fungi, viruses, and protozoa. Factors modulating microbiome abundance and composition include host genetic background, environmental parameters, and pathogen exposure. Conversely, the microbiome profoundly influences pathogen infection of the mosquito host and thus harbours considerable potential to impact the transmission of vector-borne diseases. As such, there is a growing interest in using the microbiome in novel vector-control strategies, including exploiting the natural ability of some microbes to interfere with infection of the vectors by pathogens. However, before novel microbiome-based vector control approaches can move towards translation, a more complete understanding of the interactions between mosquitoes, their microbiome, and the pathogens they transmit, is required to better appreciate how variation in the microbiome of field mosquitoes affects these interactions. To examine the impact of the host background and the associated diversity of microbiomes within distinct hosts, but without artificially manipulating the microbiome, we exposed several laboratory-reared and field-collected Aedes aegypti mosquito lines to Zika virus (ZIKV) and correlated their microbial load and composition to pathogen exposure and viral infection success. We observed significant differences in ZIKV exposure outcomes between the different mosquito lines and their associated microbiomes, and found that ZIKV alteration of the microbiomes was distinct in different lines. We also identified microbial taxa correlating with either ZIKV infection or a lack of infection. In summary, our study provides novel insights into the variability of pathogen interactions within the mosquito holobiont. A more complete understanding of which factors influence the tripartite interactions between Aedes mosquitoes, their microbiome, and arboviral pathogens, will be critical for the development of microbial-based interventions aimed at reducing vector-borne disease burden. Author summary. The mosquito microbiome composition differs within an individual across its development, as well as between individual mosquitoes at the same developmental stage, and between spatially or genomically different mosquito populations. The microbiome is highly relevant for the ability of mosquitoes to transmit pathogens. Furthermore, certain microbes have been shown to influence pathogen infection of the mosquito, while conversely, infection with a pathogen can alter the mosquito microbiome. However, we have a poor understanding how universally conserved these pathogen-related effects observed in a specific host-microbiome combination are in different mosquito populations with their respective microbiomes. To address this, we infected different mosquito lines, either reared in the laboratory or caught in the field and examined the microbiomes after exposure to Zika virus (ZIKV) compared to unchallenged microbiomes. We also examined how the virus infection progressed in different mosquito lines and correlations with further microbiome changes. The observed microbiome responses differed between host lines, potentially due to either different microbiomes associated with the respective hosts. Alternatively, the host may respond differently to the viral infection, which subsequently alters the microbiome in a distinct manner, or a combination of host and microbiome effects may occur. As microbes are being evaluated for novel approaches to control mosquito-borne disease, our findings are highly relevant to contribute to a more complete understanding of host-microbe interactions which will be critical to develop these approaches. Variation of the microbiome of different mosquito lines need to be considered in experimental designs and when interpreting results from specific studies. It is especially relevant for deployment of interventions in the field where microbial variability is known to be higher and where variation is observed between mosquito populations.

Flaviviruses have increasingly emerged and re-emerged in recent decades, infecting millions of people annually. Zika virus is particularly concerning due to its associated pathological complications, including microcephaly in newborns and Guillain-Barré syndrome in adults, posing a significant threat to public health. Despite efforts made by the scientific community, no licensed drugs against flaviviruses have been developed. Medicinal plants show promise as a novel source of antiviral agents, as they possess a diverse array of biologically active secondary metabolites, making them potential candidates for therapeutic use. We sought to investigate the antiviral potential of rosemary extract (RE) against ZIKV in human dermal fibroblasts (HFF-1 cells), one of the earliest targets of infection. ZIKV was treated with various concentrations of RE or its individual major polyphenols, including rosmarinic acid (RA), carnosic acid (CA), and carnosol (CO), and the remaining infectivity of each sample was measured by plaque reduction assay. To evaluate the impact of RE on different stages of the ZIKV replication cycle, HFF-1 cells were treated before, during, and after infection, or the virus was treated before infection. RE exerted potent antiviral activity against ZIKV in both Vero and HFF-1 cells by directly acting on virus particles before infection. Importantly, RE significantly inhibited the later stages of the virus replication cycle by interfering with post-entry mechanisms within the host cell. Moreover, major RE-derived polyphenols CA and CO, but not RA, were shown to significantly reduce ZIKV infectivity. Overall, RE significantly impairs ZIKV infection in vitro by directly interacting with virus particles prior to adsorption and interfering with post-entry processes of the ZIKV replication cycle. This study provides rigorous evidence indicating the potential development of RE as an antiviral agent, warranting further investigation into the mechanisms underlying its inhibitory activity against ZIKV and its effects on other medically important flaviviruses.

Zika virus (ZIKV) is a mosquito-borne flavivirus primarily transmitted among humans by Aedes aegypti . Over the past two decades, it has caused significant outbreaks associated with birth defects and neurological disorders. Phylogenetically, ZIKV consists of two main genotypes referred to as the African and Asian lineages, each exhibiting distinct biological properties. African lineage strains are transmitted more efficiently by mosquitoes, but pinpointing the genetic basis of this difference has remained challenging. Here, we address this question by comparing recent African and Asian strains using chimeric viruses, in which segments of the parental genomes are swapped. Our results show that the structural genes from the African strain enhance viral internalization, while the non-structural genes improve genome replication and infectious particle production in mosquito cells. In vivo mosquito transmission is most significantly influenced by the structural genes, although no single viral gene alone determines this effect. Additionally, we develop a stochastic model of in vivo viral dynamics in mosquitoes that mirrors the observed patterns, suggesting that the primary difference between the African and Asian strains lies in their ability to traverse the mosquito salivary glands. Overall, our findings suggest that the polygenic nature of ZIKV transmissibility has prevented Asian lineage strains from achieving the same epidemic potential as African lineage strains, underscoring the importance of lineage-specific adaptive landscapes in shaping ZIKV evolution and emergence.

ABSTRACT Zika virus (ZIKV) infection during pregnancy is associated with the development of fetal complications such as microcephaly. We have recently demonstrated that palmitoleate protects against ZIKV-induced apoptosis in placental trophoblasts. In the present study, we hypothesize that palmitoleate prevents ZIKV infection-induced endoplasmic reticulum (ER) stress and apoptosis in neurons. Neurons were infected with 0.1-1 multiplicity of infection of recombinant MR766 or PRVABC59 strains of ZIKV for an hour followed by treatment of palmitoleate (100 µM-200 µM) for different post-infection time points. Apoptosis was measured by nuclear morphological changes, caspase 3/7 activity, and immunoblot analysis of pro-apoptotic mediators. Activation of ER stress markers and viral envelope levels were detected using qRT-PCR and immunoblot analysis. Infectious virus particles were measured by using plaque assay. ZIKV infection to neuronal cells showed increased levels of pro-apoptotic markers like cleaved-PARP, cleaved caspase-3, Bim, and Puma, whereas decreased levels of anti-apoptotic markers such as Mcl-1, Bcl-1, and Bcl-xL. Further, we observed activation of three arms of ER stress namely: inositol requiring enzyme 1 alpha (IRE1), protein kinase-like ER kinase (PERK), and activating transcription factor (ATF6) pathways with ZIKV infection. Treatment of palmitoleate dramatically decreased ZIKV infection-induced increase in percent apoptotic nuclei and caspase 3/7 activity. Further, treatment of palmitoleate decreased cleaved PARP and PUMA protein expressions. Treatment of palmitoleate reduced ZIKV-induced ER stress activation as evidenced by decreased levels of phosphorylated forms of IRE1 and eukaryotic initiation factor 2 alpha; decreased expressions of cleaved ATF6, spliced X-box associated protein 1 and C/EBP homologous protein compared to ZIKV infection alone. Further, treatment of palmitoleate attenuated ZIKV envelope levels and infectious titer in SH-SY5Y and primary fetal cortical neurons isolated from humanized STAT2 knockin mice. These data suggest that palmitoleate supplementation protects against ZIKV-induced neuronal ER stress, apoptosis and decreases Zika viral load thereby mitigates neuronal damage.

Numerous orthoflaviviruses transmitted through the bites of different mosquito species infect more than 500 million people annually. Bite-initiated skin infection represents a critical and conserved step in transmission and a deeper understanding of this process will promote the design of broad-spectrum interventions to address diverse orthoflavivirus health threats. Here, we identify and characterize a transmission-enhancing viral factor in mosquito saliva that is shared across orthoflaviviruses. Saliva of West Nile virus-infected Culex and Zika virus-infected Aedes contains a viral non-coding RNA, subgenomic orthoflaviviral RNA (sfRNA), within lipid vesicles distinct from virions. Higher concentration of sfRNA in infectious saliva positively correlates with infection intensity in human cells and skin explants. Early sfRNA delivery into transmission-relevant skin cell types and human skin explant demonstrate that sfRNA is responsible for the infection enhancement. Co-inoculation of sfRNA in a mouse model of transmission enhanced skin infection and worsened disease severity, evidencing the role of salivary sfRNA as a transmission-enhancer. Mechanistically, salivary sfRNA attenuates early interferon response in human skin cells and skin explants by altering MDA5-mediated signaling. Our results, derived from two distinct orthoflaviviruses and supported by prior studies, establish salivary sfRNA as a pan-orthoflavivirus transmission-enhancing factor driven by a conserved viral non-coding RNA.

Vertebrate animals and many small DNA and single-stranded RNA viruses that infect vertebrates have evolved to suppress genomic CpG dinucleotides. All organisms and most viruses additionally suppress UpA dinucleotides in protein coding RNA. Synonymously recoding viral genomes to introduce CpG or UpA dinucleotides has emerged as an approach for viral attenuation and vaccine development. However, studies that investigate the effects of this recoding strategy on viral replication and pathogenesis in vivo are still limited. Flaviviruses including West Nile virus (WNV) are transmitted between vertebrate hosts by invertebrate vectors. In humans, WNV infection can cause flu-like symptoms and neuroinvasive disease. We investigated how alterations in WNV dinucleotide frequencies impact virus replication, transmission by vector mosquitoes, as well as pathogenesis and neuroinvasiveness in vertebrates. In Culex pipiens vector mosquitoes and Culex cell lines only WNV with elevated UpA frequencies displayed attenuated replication. In vertebrate cell lines and primary human neuro-astrocyte co-cultures both UpA and CpG enrichment reduced viral replication. In mice, the CpG-high WNV mutant demonstrated partial attenuation with delayed weight loss compared to wild-type WNV, though infection still resulted in 100% mortality. In contrast, 75% of animals survived inoculation with the UpA-high WNV mutant and were protected against wild-type WNV challenge. Notably, all animals that succumbed to infection had similar levels of virus in the brain, irrespective of the WNV mutant. Our results underscore the complex interplay between viral genome composition and host immune responses, highlighting potential safety concerns for dinucleotide manipulation as a strategy for live-attenuated vaccine development in flaviviruses. Importance Flaviviruses such as West Nile virus (WNV) pose significant public health concerns due to their potential to cause severe neurological disease. Synonymously recoding flavivirus genomes to introduce CpG or UpA dinucleotides has emerged as an approach for viral attenuation and vaccine development. However, the in vivo effects of manipulating these frequencies across the complete transmission cycle remained unexplored. Our study provides comprehensive insights of how CpG and UpA recoding affects WNV replication in both the mosquito vector and vertebrate hosts. We demonstrate that elevated UpA content attenuates virus replication throughout the transmission cycle, while CpG enrichment only impacts replication in the vertebrate host. Although UpA-high WNV shows significant attenuation and provides protection against wild-type infection, animals that succumb exhibit similar brain viral loads as wild-type infections. These findings have critical implications for live-attenuated vaccine development based on dinucleotide manipulation, specifically highlighting the importance of carefully evaluating the risk of neuroinvasion.

An outbreak of Zika fever occurred in Thiruvananthapuram City, Kerala, India during 2021. On request of the Kerala state health administration, we investigated the same, towards proposing requisite containment strategies of the disease outbreak. Epidemiological investigations indicated a clustering pattern of Zika fever cases with the presumed index case from a multi-speciality hospital in the city. Preliminary reports on the same had been already reported elsewhere during 2021. Further, entomological surveys carried out evinced the predominant mosquito species in the City viz., Aedes albopictus (65.55%), Aedes aegypti (22.0%) and Aedes vittatus (12.0%) were naturally infected with Zika virus (ZIKV), the Minimum Infection Rates (MIR) being 17.9, 7.8 and 3.6 respectively. Also, trans-ovarian transmission was recorded in Ae. albopictus. This is the first report on detection of ZIKV from Ae. albopictus in India. Analysis of phylogenetically informative genes of ZIKV genome indicated the emergence of a distinct lineage of the Asian strain of virus, with five unique non-synonymous mutations viz., “A22T” & “I160M” (pre-Membrane) and “D348N”, “T470A” & “V473L” (Envelope). was involved in the outbreak. The altered gene expression pattern and evolutionary implications of these unique mutations remain to be investigated. Genetic analysis of the virus isolates from this and other investigations carried out on sporadic outbreaks of ZIKV in the country subsequently, indicated ZIKV is reemerging as a distinct genetic lineage in India. These findings and other recent reports on ZIKV outbreaks warrant an urgent need for a systematic country-wide surveillance strategy, towards the prevention/ preparedness/ containment of a massive outbreak of this emerging neurovirulent arboviral disease.

Post-translational modifications play crucial roles in viral infections, yet many potential modifications remain unexplored in orthoflavivirus biology. Here we demonstrate that the UFMylation system, a post-translational modification system that catalyzes the transfer of UFM1 onto proteins, promotes infection by multiple orthoflaviviruses including dengue virus, Zika virus, West Nile virus, and yellow fever virus. We found that depletion of the UFMylation E3 ligase complex proteins UFL1 and UFBP1, as well as other UFMylation machinery components (UBA5, UFC1, and UFM1), significantly reduces infectious virion production for orthoflaviviruses but not the hepacivirus, hepatitis C. Mechanistically, UFMylation does not regulate viral RNA translation or RNA replication but instead affects a later stage of the viral lifecycle. We identified novel interactions between UFL1, and several viral proteins involved in orthoflavivirus virion assembly, including NS2A, NS2B-NS3, and Capsid. These findings establish UFMylation as a previously unrecognized post-translational modification system that promotes orthoflavivirus infection, likely through modulation of viral assembly. This work expands our understanding of the post-translational modifications that control orthoflavivirus infection and identifies new potential therapeutic targets.

ABSTRACT Zika virus (ZIKV) infection can lead to a variety of clinical outcomes, including severe congenital abnormalities. The phosphatidylserine (PS) receptors AXL and TIM-1 are recognized as critical entry factors for ZIKV in vitro . However, it remains unclear if and how ZIKV regulates these receptors during infection. In this study, we investigated AXL and TIM-1 expression in human alveolar basal epithelial A549 cells, glioblastoma U87 cells, and embryonic stem cells-derived trophoblast following ZIKV infection. We found that both the Asian strain FSS13025 and the African strain MR766 of ZIKV downregulate AXL, with a milder effect on TIM-1. We identified several ZIKV proteins, notably envelope (E), NS2A, NS3, and NS4B, that contribute to this downregulation. Notably, treatment with lysosomal inhibitor NH 4 Cl or the autophagy inhibitor 3-Methyladenine (3-MA) mitigated the AXL/TIM-1 downregulation, indicating autophagy’s involvement in the process. Importantly, this downregulation facilitates sustained viral replication and promotes viral spread by preventing superinfection and limiting cell death, which is also associated with impaired innate immune signaling. Our findings uncover a mechanism by which ZIKV downregulates entry factors to enhance prolonged viral replication and spread. AUTHOR SUMMARY Zika virus (ZIKV) infection has been associated with severe birth defects, yet the mechanisms underlying its pathogenesis remain poorly understood. In this study, we investigated phosphatidylserine (PS) receptors AXL and TIM-1 and discovered that they promote ZIKV entry but are downregulated by the virus infection. We identified several ZIKV proteins involved in AXL and TIM-1 down-regulation through an autophagy-mediated process. Mechanistically, this loss of surface receptors protects host cells from superinfection and cell death, while dampening the innate immune response, ultimately promoting viral spread. Our results contribute to a better understanding of ZIKV’s interactions with host cells and offer insight into viral entry, innate signaling, and pathogenesis.

Introduction The severity of virally induced prenatal brain injury, even among dizygotic twins, varies according to individual and maternal risk and protective factors, including genomics. Objective This scoping review aims to analyze data on genetic susceptibility to neurological outcomes in children exposed in utero to Zika virus. Methods We followed JBI methodology for this scoping review. A search in PubMed, Scopus, CINAHL, Web of Science, Academic Search Ultimate, Agricola, Health Source: Nursing/Academic Edition, and Psychology and Behavioral Sciences Collection was conducted. Three reviewers independently screened studies using the Rayyan platform. Studies on gene mutations impacting brain injury after Zika virus infection were included. Results Thirteen articles identifying candidate genes related to brain injury were reviewed. Twenty-three genes were implicated in modulating susceptibility to prenatal brain injury, including six maternal and 17 infant genes. Conclusion Maternal and fetal genetic factors likely contribute susceptibility to virally induced prenatal brain injury. Analyzing polygenic risk could aid in future screening programs to identify individuals at risk. This information may eventually be integrated into clinical data, helping healthcare providers, families, and patients understand how to personalize care for better outcomes. Impact This paper evaluates available evidence about the relationship between genetic susceptibility and neurological consequences of Zika virus exposure during pregnancy. After performing a scoping review, we identified 13 articles describing candidate genes that potentially contribute to the development of virally induced brain injury after prenatal Zika infection. Of the genes identified, six were associated with maternal risks, while 17 were linked to the fetus. Maternal and prenatal genetic factors could increase the risk of virally induced prenatal brain injury. Future research should investigate factors that can modify disease pathogenesis toward the goal of reducing the global impact of brain injury.

This study aims to describe neurological, visual, and auditory alterations in children whose mothers had confirmed Zika virus (ZIKV) infection during pregnancy, with most of these children not presenting congenital microcephaly; Methods: an observational, longitudinal, and prospective study was conducted, involving children with in utero exposure to Zika virus, following from birth up to 30 months of age; Results: of the 2,882 pregnant women admitted, 116 had a suspected ZIKV infection, of whom 33 had laboratory confirmation. Only one child presented with congenital microcephaly. Despite this, neurodevelopment delay was observed in 36.4% of children evaluated, radiological abnormalities in 29.1%, auditory abnormalities in 8.3%, and ophthalmological abnormalities in 10%; Conclusions: newborns of mothers with confirmed ZIKV infection during pregnancy may present with varying degrees of visual, auditory, and neurological impairment, despite the presence of congenital microcephaly.

This retrospective cohort study included 7,870 pregnant women (2,269 with confirmed Zika virus (ZIKV) infection and 5,601 without Zika infection) and their concepts. We obtained data from different databases in the state of Rio de Janeiro, Brazil. We used a propensity score model to control for confounding factors and stratify between the trimesters of pregnancy. Of the ZIKV+ pregnant women, 49 cases of congenital microcephaly or congenital nervous system (CNS) abnormalities were identified (2.16% or 193.9 cases in 10 000 live births), whereas 44 cases were identified among ZIKV- women (0.78% or 71.4 cases in 10 000 live births). The multivariable analysis evidenced an odds ratio of 2.46 (95% CI 1.30-4.64), 4.29 (95% CI 1.93-9.53) in the first trimester, 5.29 (95% CI 1.08-25.95) in the second trimester and 0.68 (95% CI 0.21-2.14) in the third trimester. The most common findings among ZIKV+ cases were intracranial calcifications, ventriculomegaly, posterior fossa malformations, reduced brain volume, corpus callosum malformations, cortex dysplasia, lissencephaly, and pachygyria. 55.5% of the ophthalmologic exams were abnormal, and anomalies % of brainstem auditory evoked potentials were reported in 33.3%.

ABSTRACT Zika virus (ZIKV) is an emerging arbovirus belonging to the Flaviviridae family and Orthoflavivirus genus, with a pronounced tropism for the central nervous system (CNS), where it induces neuroinflammation and neuronal death. ZIKV is known to exploit host cellular mechanisms, including the activation of survival pathways such as the PI3K/AKT signaling cascade, to evade apoptosis and enhance its replication. The phosphatidylinositol 3-kinase γ (PI3Kγ) pathway regulates critical cellular processes, including differentiation, recruitment, and survival, and is abundantly expressed in both brain tissue and leukocytes. This study aimed to investigate the role of the PI3Kγ pathway during ZIKV infection. Primary neuronal cultures from PI3Kγ-deficient mice (PI3Kγkd/kd) and human neuroblastoma SH-SY5Y cells treated with the PI3Kγ inhibitor AS605240 were infected with ZIKV to assess the impact of PI3Kγ signaling on viral replication and neuronal survival. Additionally, interferon α/β receptor knockout (A129) mice were treated with AS605240 either before or after ZIKV infection to evaluate the pathway’s role in neuroinflammation. In vitro, both genetic ablation and pharmacological inhibition of PI3Kγ suppressed ZIKV replication and prevented neuronal death. In vivo, mice treated with the PI3Kγ inhibitor exhibited enhanced protection against ZIKV infection, characterized by reduced viral load, and diminished brain and optic nerve damage. This neuroprotective effect correlated with altered astrocyte and microglia activation, marked by reduced TNF production in microglia. Furthermore, inhibition of PI3Kγ curtailed the recruitment and activation of CD8+ T cells and decreased the production of pro-inflammatory mediators, including IFN-γ and IL-17, in the brains of ZIKV-infected mice. These findings suggest that PI3Kγ activation facilitates ZIKV infection and exacerbates neuroinflammation. Pharmacological inhibition of the PI3Kγ pathway may offer therapeutic benefits by limiting viral replication and alleviating neuroinflammatory responses during ZIKV infection.

Viral genome sequencing using the ARTIC protocol has been a vital tool for understanding the spread of epidemics including Ebola, Zika, COVID-19 and Mpox and has seen widespread adoption due to its low cost and high sensitivity. Here, we describe PrimalScheme, an open-source toolkit and website that allows users to easily design primer schemes for amplicon sequencing of viruses and has generated over 67,000 primer schemes for a global community since 2017. In January 2020, PrimalScheme was used to rapidly generate a primer scheme for SARS-CoV-2, with primer pools distributed to researchers from 44 countries to help scale-up genomic surveillance efforts. Overall, these primers were used to generate an estimated 18M genome sequences and the protocols were viewed online ~250K times. To complement PrimalScheme, we have built PrimalScheme Labs, a scheme repository which allows users to find and share primers schemes as well as establishing a set of data standards. Through improvements to the primer design process, including the use of discrete primer clouds, we have expanded the use of amplicon sequencing to include diverse virus species. We demonstrate the utility of this approach through a high diversity pan-genotype Measles virus (MeV) scheme. We also demonstrate its use on a high sensitivity, short amplicon Monkeypox virus (MPXV) scheme with over 1000 primers, showing high genome recovery on low-titre clinical samples. These developments have implications for sequencing from samples such as wastewater, for genomic surveillance of endemic pathogens and in preparing for future pandemics.

Abstract Human plasma is routinely collected during clinical care and constitutes a rich source of biomarkers for diagnostics and patient stratification. Liquid chromatography-mass spectrometry (LC-MS)-based proteomics is a key method for plasma biomarker discovery, but the high dynamic range of plasma proteins poses significant challenges for MS analysis and data processing. To benchmark the quantitative performance of neat plasma analysis, we generated a multispecies sample set based on a human tryptic plasma digest containing varying low level spike-ins of yeast and E. coli tryptic proteome digests, termed PYE. The sample set was distributed across twelve different sites and analysed on state-of-the-art LC-MS platforms in data-dependent (DDA) and data-independent acquisition (DIA) modes, resulting in a total of 1,116 individual LC-MS runs. Centralized data analysis showed that DIA methods outperform DDA-based approaches regarding identifications, data completeness, accuracy, and precision. DIA achieved excellent technical reproducibility, as demonstrated by coefficients of variation (CVs) between 1.5% and 4.6% at protein level. Comparative analysis of different setups clearly shows a high overlap in identified proteins and proves that accurate and precise quantitative measurements are feasible across multiple sites, even in a complex matrix such as plasma, using state-of-the-art instrumentation. The collected dataset, including the PYE sample set and strategy presented, serves as a valuable resource for optimizing the accuracy and reproducibility of LC-MS and bioinformatic workflows for clinical plasma proteome analysis.

Dengue fever, caused by the dengue virus (DENV), presents a significant global health challenge, especially in tropical and subtropical regions. The exponential increase in native DENV cases in Europe is concerning, indicating a significant rise in incidence. As climate change intensifies, it is crucial to implement proactive measures to address the presence of Aedes albopictus (Asian tiger mosquito) in Europe to prevent dengue and other tropical diseases from becoming endemic in temperate regions. In 2024, Brazil experienced a dramatic surge in dengue cases, reporting an incidence rate four times higher than that of 2023, accompanied by a notable rise in mortality. This alarming trend underscores the urgent need for heightened surveillance and preventive measures, as it may signal the potential for new dengue outbreaks in non-tropical regions. Herein, we explored various aspects of DENV transmission, clinical manifestations, epidemiological trends, and management strategies. DENV transmission depends on Aedes mosquitoes as vectors and humans as hosts. The Aedes species is easily identified by its distinctive black-and-white striped legs. Factors such as temperature, humidity, and precipitation significantly influence mosquito proliferation, and these factors are increasingly affected by climate change. Additionally, the rise in global travel is facilitating the spread of viruses across borders, particularly arboviruses like DENV. Understanding these dynamics is crucial for effective public health interventions. DENV presents a spectrum of clinical manifestations, ranging from asymptomatic cases to severe conditions such as dengue hemorrhagic fever and dengue shock syndrome, influenced by viral serotype and host factors. We explored various diagnostic approaches that integrate clinical symptoms with serological and molecular testing. In Brazil, prevention strategies including vector control, community engagement, and vaccination programs have been implemented. However, these measures have proven insufficient to avert the outbreak in 2024. Climate change drives the geographical expansion of dengue-endemic areas, with recent outbreaks in Europe highlighting the evolving epidemiological landscape. Vigilant global collaboration is essential to mitigate the impact of dengue and other emerging arboviruses, including West Nile virus, Zika virus, Chikungunya virus, Oropouche virus, and Yellow Fever virus.

Abstract Zika virus (ZIKV) poses a significant threat due to its association with severe neurological complications, particularly during pregnancy. Although viruses exhibit tropism for neural cells, including astrocytes, the role of these cells in controlling ZIKV replication remains unclear. In this study, we demonstrated that ZIKV induces caspase-1 activation in primary astrocytes despite the absence of classical signs of inflammasome activation. Caspase-1/11 -/- astrocytes exhibit heightened permissiveness to viral replication, accompanied by overactivation of glycolytic metabolism. Inhibition of glycolysis reversed the susceptibility of caspase-1/11 -/- astrocytes to ZIKV infection. Protein network analysis revealed mTORC as a link between proteins involved in glycolysis and caspase-1, and mTORC inhibition also suppressed viral replication. Furthermore, we found that the impact of caspase-1/11 on astrocytes depends on the regulation of pyruvate transport to mitochondria for viral replication. Overall, our findings elucidate a caspase-1/11-dependent microbicidal mechanism in astrocytes that involves the mTORC/glycolytic pathway/pyruvate axis, providing insights into potential therapeutic targets for ZIKV infection.

Several RNA viruses induce widespread degradation of cellular mRNAs upon infection; however, the biological significance and mechanistic details of this phenomenon remain unknown. Here, we make use of a model alphavirus, Sindbis virus (SINV), to fill this knowledge gap. We found that SINV triggers cellular RNA decay through the exonuclease XRN1 and the 5’-to-3’ degradation machinery (5-3DM). These proteins accumulate at viral replication organelles (VROs) and interact with the non-structural protein 1 (nsP1), bringing mRNA degradation into proximity with vRNA synthesis. Our data suggest that monophosphate nucleotides released by cellular RNA decay are recycled through the salvage pathway to feed viral replications. Our work thus reveals a fundamental connection between cellular mRNA degradation and viral replication via nucleotides repurposing. Research highlights 5’-3’ RNA decay is essential for the replication of a wide range of viruses. XRN1 directly interacts with transcripts which are degraded during infection. RNA decay factors and salvage pathway members localise to viral factories. Supplying nucleosides to several 5-3DM deficient cells facilitates SINV infection.

The chemical complexity of natural products such as Eugenia punicifolia (Kunth) DC. plant, presents a challenge when extracting and identifying bioactive compounds. This study investigates the impact of different extraction systems and seasonal variations on the chemical profile and pharmacological potential of E. punicifolia leaves using NMR spectroscopy for chemical analysis and canonical correlation analysis (CCA) for bioactivity correlation. Extracts obtained with methanol (M), ethanol (E), methanol:ethanol [1:1 (ME)], and methanol:ethanol:water [3:1:1 (MEW)] were analyzed for antioxidant, antiglycation and antiviral activities. Quantitative ¹H NMR, combined with the PULCON method, was used to quantify phenolic compounds such as quercetin, myricetin, catechin and gallic acid. The results showed that the MEW extract obtained in the rainy season exhibited the highest antioxidant and antiglycation activities, with an AGE inhibition capacity greater than 93%. Furthermore, our results showed that all the extracts were able to inhibit over 94% of the Zika virus (ZIKV) infection in Vero E6 cells. The CCA established strong correlations between the phenolic compounds and bioactivities, identifying gallic acid, catechin, quercetin and myricetin as key chemical markers. This study demonstrates the importance of selecting appropriate extraction systems and considering seasonality to optimize the pharmacological potential of E. punicifolia leaves and highlights the efficacy of NMR in linking chemical composition with bioactivities.

Zika fever (ZIKAF) is an emerging mosquito-borne flavivirus illness of humans. The etiological agent, ZIKA virus (ZIKAV), though known to be distributed in the tropics causing sporadic cases, its rapid global expansion with pandemic potential raised global concern. Abrupt emergence in South American countries, Caribbean, and the Americas, the WHO declared ZIKA a public health emergency of international concern in 2016. ZIKAV usually causes mild infections, however, its recent unusual presentations of Guillen–Barré syndrome in adult and microcephaly in newborn babies of ZIKAV-infected mothers in Brazil alerted global public health authorities. Certain mutations on virus genome have been found correlating with clinical severity and its unusual transmission routes through sexual and blood transfusions emphasizes the necessity for understanding the virological determinants and its impact. Abrupt re-emergence in India (2018–2019) particularly in Gujarat (2016), Tamil Nadu (2017), Uttar Pradesh (2021), Maharashtra, Kerala (2021), and Karnataka (2023), indicate urgent measures for strengthening surveillance systems for designing effective prevention and control measures in the country. Given the global concern, here we reviewed the current knowledge of global ZIKAV genetic lineages vis-a-vis the Indian scenario and discusses the future priorities in ZIKAV research in India for effectively designing control strategies.

The Flaviviridae are a family of viruses that include the important arthropod-borne human pathogens dengue virus (DENV), West Nile virus, Zika virus, Japanese encephalitis virus, and yellow fever virus. Flavivirus nonstructural protein 1 (NS1) is essential for virus replication but is also secreted from virus-infected cells. Extracellular NS1 acts as a virulence factor during flavivirus infection in multiple ways, including triggering endothelial dysfunction and vascular leak via interaction with endothelial cells. While the role of NS1 in inducing vascular leak and exacerbating pathogenesis is well appreciated, if and how NS1-triggered endothelial dysfunction promotes virus infection remains obscure. Flaviviruses have a common need to disseminate from circulation into specific tissues where virus-permissive cells reside. Tissue-specific dissemination is associated with disease manifestations of a given flavivirus, but mechanisms dictating virus dissemination are unclear. Here we show that NS1-mediated endothelial dysfunction promotes virus dissemination in vitro and in vivo . In mouse models of DENV infection, we show that anti-NS1 antibodies decrease virus dissemination, while the addition of exogenous NS1 promotes virus dissemination. Using an in vitro system, we show that NS1 promotes virus dissemination in two distinct ways: (1) promoting crossing of barriers and (2) increasing infectivity of target cells in a tissue- and virus-specific manner. The capacity of NS1 to modulate infectivity correlates with a physical association between virions and NS1, suggesting a potential NS1-virion interaction. Taken together, our study indicates that flavivirus NS1 promotes virus dissemination across endothelial barriers, providing an evolutionary basis for virus-triggered vascular leak. Author Summary The Flaviviridae contain numerous medically important human pathogens that cause potentially life-threatening infections. Over half of the world’s population is at risk of flavivirus infection, and this number is expected to increase as climate change expands the habitats of the arthropod vectors that transmit these flaviviruses. There are few effective vaccines and no therapeutics approved for prevention or treatment of flavivirus infection, respectively. Given these challenges, understanding how and why flaviviruses cause pathogenesis is critical for identifying targets for therapeutic intervention. The secreted nonstructural protein 1 (NS1) of flaviviruses is a conserved virulence factor that triggers endothelial dysfunction in a tissue-specific manner. It is unknown if this endothelial dysfunction provides any benefit for virus infection. Here we provide evidence that NS1-triggered endothelial dysfunction facilitates virus crossing of endothelial barriers and augments infection of target cells in vitro and promotes virus dissemination in vivo . This study provides an evolutionary explanation for flaviviruses evolving the capacity to trigger barrier dysfunction and highlights NS1 and the pathways governing endothelial dysfunction, as therapeutic targets to prevent flavivirus dissemination.

Abstract Curative drugs are needed for the treatment of viral infections. Small interfering (si)RNA offer such a prospect but require the development of safe, effective and non-hepatotropic subcellular delivery systems. Here, 5 candidate siRNA molecules targeting defined sequences within the Zika virus (ZIKV) genome were assayed for their ability to reduce ZIKV induced cytopathic effects in vitro . The protection of Huh7 cells from ZIKV cytopathic effects was recorded after electroporation and the siRNA Feron-Zv2, resulted in 122.7 ± 5.3% cell viability (n = 3 ± standard error of the mean (SEM), 100nM siRNA) after exposure to ZIKV relative to a virus treated control (35.2 ± 7.1% cell viability (n = 3 ± SEM)). Protection of BHK cells was recorded after transfection with an attenuated anthrax toxin containing an RNA binding domain. Treatment with Feron-Zv4, resulted in 75.1 ± 2.9% cell viability (n = 3 ± SEM, 25nM siRNA) after exposure to ZIKV. This protection was mirrored by a system containing octameric PA where a maximum of 86.2 ± 4.4% cell viability was reported (n = 3 ± SEM, 75nM siRNA)) after treatment with Feron-Zv2. Scrambled siRNA afforded no measurable protection. Here we report for the first time that siRNA delivered by either attenuated anthrax toxin or octamer forming ATx can protect mammalian cells from ZIKV cytopathic effects.