Abstract Bats are reservoirs of emerging zoonotic viruses of concern that cause severe disease in humans and agricultural animals. However, it is poorly understood how bats are able to tolerate diverse viral infections, knowledge that could help pave the way for new therapeutic strategies. Here, we characterized antiviral pathways in two divergent bat species, Pteropus alecto and Eptesicus fuscus, identifying unique bat-specific mechanisms underlying their enhanced antiviral tolerance. We demonstrate the critical roles of STAT1 and STAT2 in IFNβ signaling, along with species-specific adaptations that collectively contribute towards a “steady and ready” antiviral state in bat cells. Unlike in humans, we find that bat interferon signalling processes resist the immune antagonistic properties of viruses like MERS-CoV which further explains the ability of bats to tolerate coronavirus infections. Using transcriptomic analysis, we identified canonical and non-canonical interferon stimulated genes (ISGs) including two key bat genes, IFIT1 and GBP1. Compared to their human orthologs, we show that bat IFIT1 and GBP1 exhibit enhanced antiviral activity against a wide range of RNA and DNA viruses, including coronaviruses and additional bat-derived poxviruses (e.g., Eptesipoxvirus). Ultimately, our work provides important insights into the evolution of enhanced interferon-mediated antiviral responses in bats, contributing to their unique ability to resist viral diseases.

West Africa serves as a critical region for the co-circulation of mosquito-borne flaviviruses, which often precipitate sporadic outbreaks. This study investigated the seroprevalence of dengue virus serotypes 1-4 (DENV-1-4), Zika virus (ZIKV), yellow fever virus (YFV), and West Nile virus (WNV) in three regions of Senegal: Sindia, Thies, and Kedougou. We retrospectively analyzed 470 serum samples for flavivirus immunoglobulin G (IgG) using a DENV-2 envelope (E) ELISA. Our findings revealed a seroprevalence of 37.23% for DENV-2 E IgG, indicative of a prior flavivirus exposure rate. The IgG seroprevalence rates for DENV-1-4, ZIKV, YFV, or WNV NS1 were 57.14%, 12.57%, 80.57% and 17.14%, respectively, with 72% of individuals harboring neutralizing antibodies against two or more flaviviruses. We also identified that residing in Sindia (ZIKV, OR, 9.428; 95% CI: 1.882-47.223 & WNV, OR, 6.039; 95% CI: 1.855-19.658) and Kedougou (ZIKV, OR, 7.487; 95% CI: 1.658-33.808 & WNV, OR, 1.142; 95% CI: 0.412-3.164) was a significant risk factor for ZIKV and WNV exposure. In contrast, history of malaria significantly reduced the risk of WNV exposure (aOR, 0.402; 95% CI: 0.203-0.794). This study underscores the complexity of flavivirus epidemiology in West Africa and the necessity for enhanced surveillance to inform public health strategies.

Zika virus (ZIKV), belonging to the Flaviviridae family, has been a severe threat to human health since the worldwide outbreak. ZIKV is capable of inducing fetal microcephaly, Guillain-Barré syndrome, and other serious neurological complications. Polypyrimidine tract-binding protein 1 (PTBP1) is a key member of the heterogeneous nuclear ribonucleoproteins (hnRNPs) family, functioning in selective mRNA splicing and gene expression regulation. Our previous study has indicated that the expression of PTBP1 increases in astrocytes upon ZIKV infection, yet the precise regulatory mechanisms underlying its role in viral replication remain elusive. In this study, we elucidated the specific pathway by which ZIKV upregulates PTBP1 expression through the activation of Hypoxia-inducible factor-1α (HIF-1α) expression. Further investigation revealed that overexpression of PTBP1 effectively inhibits viral replication, whereas knockdown of PTBP1 significantly enhances viral replication. Mechanistically, using co-immunoprecipitation assays for protein interaction screening, we identified an interaction between PTBP1 and ZIKV non-structural protein NS1. Detailed studies demonstrated that PTBP1 bound and colocalized with NS1 to lead to the degradation of NS1 protein via a lysosomal pathway. Collectively, our findings unveil a novel mechanism underlying that ZIKV infection induces the expression of PTBP1 via the HIF-1α pathway, subsequently the accumulated PTBP1 binds to ZIKV NS1 protein to promote NS1 degradation, thereby effectively inhibiting viral replication. The study illustrates a distinct restricted cellular factor that regulates ZIKV replication, which provides a potential target for the control of the viral replication and pathogenesis during the ZIKV epidemic. Importance Since the outbreak of ZIKV infection among human in 2014, a Zika epidemic has caused Zika fever accompanied with fetal microcephaly, Guillain-Barré syndrome, and other neurological symptoms. Emerging evidence reveals that ZIKV infects astrocytes to specially induce the expression of polypyrimidine tract-binding protein 1 (PTBP1), one of hnRNPs members. However, the interplay between PTBP1 and ZIKV replication is highly concerned. Here, we uncover a distinct manner that ZIKV infection induces PTBP1 expression through the activation of hypoxia-inducible factor-1α (HIF-1α) signal. Additionally, activation of HIF-1α signal hinders ZIKV replication relying on PTBP1 accumulation. Further investigations suggest that PTBP1 restrains ZIKV replication by interacting with ZIKV NS1 protein, thereby leading to the degradation of NS1 protein via a lysosomal pathway. Collectively, our findings illustrate a novel restricted cellular factor PTBP1 mediated by HIF-1α that regulates ZIKV replication, which provides a potential therapeutic target of the viral replication and pathogenesis against ZIKV pandemic.

Background Flavivirus infections pose a significant global health burden, highlighting the need for safe and effective vaccination strategies. Co-administration of different vaccines, including licensed flavivirus vaccines, is commonly practiced providing protection against multiple pathogens while also saving time and reducing visits to healthcare units. However, how co-administration of different flavivirus vaccines de facto affects immunogenicity, particularly with respect to T cell responses, is only partially understood. Methods and findings Antigen-specific T cell responses were assessed in study participants enrolled in a previously conducted open-label, non-randomized clinical trial. In the trial, vaccines against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), or yellow fever virus (YFV) were administered either individually or concomitantly in different combinations in healthy study participants. Peripheral blood samples were collected before vaccination and at multiple time points afterward. To analyze antigen-specific CD4+ and CD8+ T cell responses, PBMCs were stimulated with overlapping peptide pools from TBEV, JEV, YFV, and Zika virus (ZIKV) envelope (E), capsid (C), and non- structural protein 5 (NS5) viral antigens. A flow cytometry-based activation-induced marker (AIM) assay was used to quantify antigen-specific T cell responses. The results revealed remarkably similar frequencies of CD4+ and CD8+ T cell responses, regardless of whether vaccines were administered individually or concomitantly. In addition, administering the vaccines in the same or different upper arms did not markedly affect T cell responses. Finally, no significant cross-reactivity was observed between TBEV, JEV, and YFV vaccines, nor with related ZIKV-specific antigens. Conclusions TBEV or JEV vaccines can be co-administered with the live attenuated YFV vaccine without any markedly altered antigen-specific CD4+ and CD8+ T cell responses to the respective flaviviruses. Additionally, the vaccines can be delivered in the same or different upper arms without any significant influence on the T cell response. From a broader perspective, these results provide valuable insights into the outcome of immune responses following simultaneous administration of different vaccines for different but related pathogens. Author summary Why was this study done? The World Health Organization recently declared a global initiative to control arboviral diseases. Many of these are caused by pathogenic flaviviruses, most transmitted by mosquitos and other arthropod vectors such as ticks. Vaccination is a key intervention for diseases caused by flaviviruses. Co-administration of different vaccines, including currently licensed flavivirus vaccines, is commonly practiced. Co-administration of vaccines saves time and reduces the number of visits to healthcare facilities and vaccine clinics. Cellular immune responses have not been thoroughly evaluated upon co- administration of currently licensed flavivirus vaccines, including yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), and Japanese encephalitis virus (JEV) vaccines. What did the researchers find? The magnitude and specificity of CD4+ and CD8+ T cell responses to virus- specific antigens remained largely unchanged by the concomitant delivery of the studied flavivirus vaccines. Concomitant delivery of vaccines in the same or different upper arms of the study participants had minimal impact on CD4+ and CD8+ T cell responses. The studied vaccines maintained distinct CD4+ and CD8+ T cell reactivity across their respective viral antigens without generating any significantly detectable cross-reactivity to each other or ZIKV-antigens. What do these findings mean? Along with recently published data from the present study cohort, co- administration of three commonly used current licensed flavivirus vaccines is feasible without increasing the risk of adverse events or significantly affecting the development of either neutralizing antibodies (nAbs) or T cell responses against the respective viral antigens.

The vast, untapped potential of the world's oceans is revealing groundbreaking advancements in human health and vaccination. Marine microalgae such as Nannochloropsis spp. and Dunaliella salina are emerging as vital sources for recombinant vaccine development. Various specific and heterologous genetic tools have been generated for Dunaliella salina and Nannochloropsis spp. To boost production of functional recombinant antigens and even the induction of immunoprotective responses. In humans, promising antigens produced in microalgae have shown potential in combating diseases caused by the Human papillomavirus, Human immunodeficiency virus, Hepatitis B virus, Influenza virus, Zika virus, Zaire Ebola virus, Plasmodium falciparum, and Staphylococcus aureus. For animals, microalgae-derived vaccine prototypes have been developed to fight against the Foot and mouth disease virus, Classical swine fever virus, Vibriosis, White spot syndrome virus, and Histophilus somni. Compared to the sources of other drugs, marine organisms offer unique advantages, including the ability to express complex antigens and sustainable production. Additionally, the ocean provides an array of bioactive compounds that serve as therapeutics, potent adjuvants, delivery systems, and immunomodulatory agents. These innovations from the sea not only enhance vaccine efficacy but also contribute to broader immunological and general health. This review explores the transformative role of marine-derived substances in modern medicine, emphasizing their importance in the ongoing battle against infectious diseases.

Abstract Though generally mild in humans, Zika virus (ZIKV) frequently causes severe disease and congenital defects in children born to mothers infected during pregnancy (Musso et al. NEJM 2019 PMID: 31597021; Shan et al. Cell Host Microbe 2018 PMID: 30008291). Previously, chimeric candidate vaccine YF-ZIK was shown to protect mice against lethal ZIKV infection and congenital malformations after single-dose immunization (Kum et al. npj Vaccines 2018 PMID: 30564463; Kum et al. Emerg. Microbes Infect. 2020 PMID: 32116148). Here we demonstrate that YF-ZIK is safe, induces strong antiviral immunity, and protects rhesus macaques against vigorous experimental challenge. YF-ZIK triggered within 7-14 days after a single subcutaneous dose high levels of neutralizing antibodies, which could significantly be boosted by a second immunization 4 weeks later. Passively transferred immune serum protects AG129 mice from lethal ZIKV challenge, supporting neutralizing antibodies as major correlate of protection. Humoral immunity is complemented by ZIKV-specific cellular responses, characterized by a balanced Th1/Th2 cytokine production. Transcriptional analyses demonstrate the induction of a gene expression profile that is comparable to that of the original yellow fever YF17D vaccine, involving multiple pathways favoring polyvalent immunity. When YF-ZIK vaccinated macaques were challenged with a high intradermal dose of ZIKV, viral RNA remains undetectable in most animals. Immunity may be sterilizing immunity as no elevation of ZIKV neutralizing antibodies, nor seroconversion to anti-ZIKV NS1 antibodies was evident. Protection is associated with an activation of innate and adaptive immunity early after challenge. Systems analysis unveils B-cell receptor TNFRSF17 as predictor for strong antibody responses to YF-ZIK vaccination in common with YF17D (Gaucher et al. J. Exp. Med. 2008 PMID: 19047440; Querec et al. Nat. Immunol. 2009 PMID: 19029902; Fourati et al. Nat. Immunol. 2022 PMID: 36316476 ), along with GNAS and CD207 (Langerin) as novel biomarkers predicting clinical outcomes. The favorable preclinical safety, immunogenicity and efficacy of YF-ZIK justifies future evaluation in humans.

Rising global temperatures coupled with increasing international travel, and trade are contributing to spread of vectors such as ticks and mosquitoes, resulting in a surge of vector-borne flavivirus infection in human population. Furthermore, this increase in flavivirus infection pose threat to the safety of biologics such as cell and gene therapy products as human-derived materials are commonly used during manufacturing of these drug products. In this study, we conducted time-course transcriptomic and protein analyses to uncover the host molecular factors driving the virulence of Zika virus (ZIKV) and Dengue virus (DENV) in the context of host defense mechanisms, as these two viruses have caused the most recent and significant flavivirus outbreaks. Compared to DENV, ZIKV exhibited stronger virulence and cytopathic effects. RNA sequencing analysis revealed differential expression of various cellular factors, including RNA processing factors. Further investigation identified cell-type and time-dependent upregulation nonsense-mediated RNA decay (NMD), RNA degradation factors and nuclear pore complex (NPC) transcripts. Protein analysis showed that ZIKV, unlike DENV, degrades NMD factors in host cells, which along with mis-regulation of RNA degradation factors resulted in accumulation in host intronic transcripts as revealed by RNA-seq data. We also found that active nuclear transport is required for ZIKV replication, suggesting that therapeutic targeting of the NPC could potentially be effective in controlling ZIKV infection. Furthermore, from our findings we hypothesize that, ZIKV, but not DENV, drives early host cell cytopathy through targeted protein degradation. Current studies are underway to develop novel strategies to detect ZIKV, DENV and other flaviviruses in biologics based on transcriptomics and proteomics. Teaser Exploring the molecular basis of flavivirus virulence in host cells.

Abstract Purpose This study aimed to evaluate the association between microcephaly and hearing disorders in children with exposed or suspected of exposure to Zika virus (ZIKV) during the intrauterine period. Methods This cross-sectional study, we enrolled children exposed or suspected of being exposed to ZIKV and born to mothers with confirmed or suspected ZIKV infection during pregnancy, admitted to the hospital between April 2016 and July 2018, and followed up until September 2021. All children underwent at least one automated auditory brainstem response (AABR) test. For analysis, the patients were divided into four groups: those with microcephaly, without microcephaly, suspected ZIKV infection, and controls. Other causes of microcephaly were excluded. Hearing impairment was assessed using the AABR to determine associations with microcephaly or central nervous system (CNS) abnormalities. Results Of the 134 children included, 34 (25.4%) were diagnosed with congenital Zika syndrome (CZS), of whom 28 (82.4%) had microcephaly, and the remaining six (17.6%) without microcephaly. Among the 28 children with microcephaly, 3 (10.7%) had abnormal AABR. Among children without microcephaly (n = 106), 3 (2.8%) had abnormal AABR (p = 0.09). Conclusion In our study population, children with and without microcephaly had abnormal AABR.

ABSTRACT Small molecules targeting specific RNA binding sites, including stable and transient RNA structures, are emerging as effective pharmacological approaches for modulating gene expression. However, little is understood about how stable RNA secondary structures are shared across organisms, an important factor in controlling drug selectivity. In this study, I provide an analytical pipeline named RNA Secondary Structure Finder (R2S-Finder) to discover short, stable RNA structural motifs for humans, Escherichia coli ( E. coli ), SARS-CoV-2, and Zika virus by leveraging existing in vivo and in vitro genome-wide chemical RNA-probing datasets. I found several common features across organisms. For example, apart from the well-documented tetraloops, AU-rich tetraloops are widely present in different organisms. I also found that the 5’ untranslated region (UTR) contains a higher proportion of stable structures than the coding sequences in humans, SARS-CoV-2, and Zika virus. In general, stable structures predicted from in vitro (protein-free) and in vivo datasets are consistent in humans, E. coli , and SARS-CoV-2, indicating that most stable structure formation were driven by RNA folding alone, while a larger variation was found between in vitro and in vivo data with certain RNA types, such as human long intergenic non-coding RNAs (lincRNAs). Finally, I predicted stable three- and four-way RNA junctions that exist both in vivo and in vitro conditions, which can potentially serve as drug targets. All results of stable sequences, stem-loops, internal loops, bulges, and three- and four-way junctions have been collated in the R2S-Finder database ( https://github.com/JingxinWangLab/R2S-Finder ), which is coded in hyperlinked HTML pages and tabulated in CSV files.

ABSTRACT Background Viruses such as dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) pose major threats to human health, causing endemic, emerging, and reemerging diseases. These arboviruses have complex life cycles involving Aedes mosquitoes, driven by environmental, ecological, socioeconomic, and cultural factors. In Colombia, Aedes aegypti is the primary vector, but Aedes albopictus is expanding across the country. Understanding the unique characteristics of each species is crucial for managing arbovirus spread, particularly in areas where they coexist. Methodology/Principal Findings We conducted an entomological survey of A. aegypti and A. albopictus (larvae, pupae, and adults) in urban and rural areas of four municipalities across different elevations (200–2200 meters above sea level) in Colombia. Household conditions and knowledge of DENV were assessed through interviews. Female A. albopictus were tested individually for arbovirus RNA, while A. aegypti were tested in pools. Both species were found up to 2100 masl. A. aegypti comprised 78% of the immature forms collected, while A. albopictus made up 22%. Larvae from both species coexisted in common artificial breeding sites in urban and rural areas, with no evidence of competition. A. albopictus preferred rural areas, lower elevations ( 270 mm), and poor household conditions, while A. aegypti was more abundant in urban areas, intradomicile environments, and areas with moderate precipitation (100–400 mm). Biting risk was higher for A. aegypti (0.02–0.22 females per person), particularly in urban areas, while A. albopictus exhibited lower biting risk (0.001–0.08), with the highest values in rural Patía. Natural infections of DENV (12.4%) and CHIKV (12.4%) were found in A. aegypti , while A. albopictus showed CHIKV (41.2%) and DENV (23%) infections, with virus dissemination to the legs and salivary glands. Conclusions/Significance Integrating household conditions and community knowledge with environmental data can enhance predictive models for vector presence and guide surveillance and educational strategies. Our findings highlight the need to consider A. albopictus as a potentially significant arbovirus vector in Colombia, especially given the presence of arboviruses in its salivary glands, its use of artificial breeding sites, its biting risk inside homes, and its differing ecological preferences and seasonal associations compared to A. aegypti . AUTHOR SUMMARY In this study, we investigated the ecological and epidemiological dynamics of Aedes aegypti and Aedes albopictus and their natural infection with DENV, ZIKV, and CHIKV in Colombia. An entomological survey conducted across four municipalities revealed distinct environmental and human-related factors influencing the distribution and abundance of these species. A. aegypti was more abundant in urban environments, favoring areas with lowest precipitation and a range of household conditions, while A. albopictus was more common in rural areas with higher precipitation and poorer household conditions. Both species were naturally infected with DENV and CHIKV, with A. albopictus showing the ability to disseminate, as indicated by their presence in the legs and salivary glands. Our findings underscore the importance of understanding species-specific ecological characteristics and incorporating community knowledge into predictive models to improve vector surveillance and control strategies, particularly in regions where both species coexist and contribute to arbovirus transmission.

Background During the 2023-dengue outbreak in Bangladesh, a diagnostic evaluation study was conducted to investigate concurrent Zika virus (ZIKV) and dengue virus (DENV) transmission in Dhaka in 2023. Aims The study explored to simultaneously detect the presence of ZIKV, DENV, and/or CHIKV while considering relevant clinical and epidemiological risk factors, using a real-time multiplex RT-PCR system. Following this, it was planned to sequence the selected samples to identify genetic variations of the ZIKV infections within the population. Methods This study was designed as a diagnostic evaluation, where participants meeting the inclusion criteria were prospectively recruited with written informed consent. A total of 399 febrile individuals were screened, with 185 meeting the inclusion criteria of having a fever onset within 2 – 5 days, along with one of the following clinical features, e.g. headache, myalgia, arthralgia or bone pain, rash, nausea, vomiting, or diarrhea and 152 undergoing real-time RT-PCR testing. Results Five ZIKV-positive cases were identified, including one DENV-ZIKV co-infection. Phylogenetic analysis revealed the ZIKV strains were part of the Asian lineage, closely related to Cambodian and Chinese strains from 2019. All ZIKV-positive cases were male, residing within a one-kilometer radius, with no prior travel history, suggesting community-level transmission. Conclusion This study marks the first identification of ZIKV in Dhaka city and the first report of ZIKV-DENV co-infection in Bangladesh that highlights the diagnostic challenges posed by the symptomatic similarities between ZIKV and other arboviruses and underscores the need for enhanced surveillance and public health interventions to mitigate the spread and impact of ZIKV in dengue-endemic regions. Synopsis This study explores the co-transmission of Zika (ZIKV) and dengue (DENV) during the 2023 dengue outbreak in Bangladesh, shedding light on important public health and epidemiological issues. While Zika is typically a mild illness for most people, it can have serious neurological consequences, such as microcephaly in infants. Dengue, another mosquito-borne virus common in tropical regions like Bangladesh, affects a significant portion of the population. The research stands out because it marks the first recorded Zika cases in Dhaka and the first instance of Zika-Dengue co-infection in the country. This discovery is crucial for both local and global health communities, as it highlights the challenge of managing outbreaks of similar viruses at the same time, complicating diagnosis and treatment. The study calls for stronger surveillance and better public health measures to manage these viruses, particularly in densely populated areas where mosquito-borne illnesses spread rapidly. This work focuses on the importance of raising awareness and enhancing disease management practices to reduce the risks posed by these infections. The overlap in symptoms and the potentially serious outcomes for vulnerable groups, like pregnant women and newborns, make understanding Zika’s transmission and genetic variations critical. These insights can guide future efforts in vaccine development and public health interventions at the community level.

Musashi RNA binding proteins are important post-transcriptional regulators of stem cell homeostasis and are known to be involved in viral infections. However, their role in SARS-CoV-2 infection remains largely unknown. Using computational studies, in vivo RNA immunoprecipitation and biochemical assays, here, we establish that Musashi 1 (Msi1) interacts with viral genomic RNA through direct binding to the SARS-CoV-2 3'UTR. Importantly, binding of Msi1 to the viral 3'UTR results in translational repression mediated by inhibition of Poly (A) binding protein (PABP). Conversely, Msi1 knockout promotes robust viral replication and increased viral protein expression. Using 2D cell cultures, stem cells and 3D organoids, we show that depletion of Msi1 in intestinal cells augments infection. This finding explains why the human intestine serves as a reservoir for the SARS-CoV-2 virus, wherein differentiated enterocytes, which have negligible levels of Msi1, are highly affected. Contrarily, stem cells which are enriched for Msi1 expression, are known to be less permissive to SARS-CoV-2 infection despite expressing the entry receptors. Our findings show how translation repression of SARS-CoV-2 by stem cell RNA binding proteins such as Msi1 could help evasion of infection.

Teicoplanin (TP) is a glycopeptide antibiotic used for Gram-positive bacterial infections, and it has been reported to inhibit SARS-CoV-2 and Ebola virus entry through cathepsin inhibition. Given that TP can inhibit viruses belonging to different virus families, we aimed to expand the potential targets of TP to determine whether TP can be developed as a broad-spectrum antiviral agent. Considering the original indication of TP, we first determined the effects of TP against viruses that cause respiratory tract infections and found that TP inhibits enveloped and non-enveloped RNA viruses, namely: human and avian influenza viruses; representative coronaviruses including porcine epidemic diarrhea virus (PEDV), human coronavirus OC43 (HCoV-OC43), and SARS-CoV-2; measles virus; human respiratory syncytial virus A2; and enterovirus 71 (EV-71). Representative flaviviruses, Zika virus (ZIKV) and dengue virus serotype 2 (DENV2), were also susceptible to inhibition by TP. In contrast, TP did not attenuate infection of human adenovirus 5, a non-enveloped DNA virus. Addition of TP at the endocytosis stage but not at the attachment/binding stage of PEDV infection reduced PEDV production in vitro , indicating cathepsin inhibition. Meanwhile, addition of TP during either the attachment/binding or the endocytosis stage of ZIKV infection reduced ZIKV particle production in host cells, and in silico modeling suggested that TP has potential binding pockets in the envelope proteins of ZIKV and DENV2. These results show that TP can be developed as a broad-spectrum antiviral especially against RNA viruses, with potentially different targets in the replication cycle of various viruses.

Summary Zika virus (ZIKV) infection during pregnancy can lead to fetal brain infection and developmental anomalies collectively known as congenital Zika syndrome (CZS). To define the molecular features underlying CZS in a relevant human cell model, we evaluated ZIKV infection and neurodevelopment in primary fetal brain explants and induced pluripotent stem cell-derived mixed neural cultures at single cell resolution. We identified astrocytes as key innate immune sentinel cells detecting ZIKV and producing IFN-β. In contrast, neural progenitor cells displayed impaired innate immunity and supported high levels of viral replication. ZIKV infection of neurons suppressed differentiation and synaptic signaling networks and programmed a molecular switch from neurogenesis to astrogliogenesis. We identified a universal ZIKV-driven cellular stress response linked to intrinsic apoptosis and regulated by IFN-β. These findings reveal how innate immune signaling intersects with ZIKV-driven perturbations in cellular function to influence CZS outcomes including neuron developmental dysfunction and apoptotic cell death.

Despite decades of research, survival from brain cancer has scarcely improved and is drastically lower than that of other cancers. Novel therapies, such as immunotherapy, hold great promise for treating brain tumours and are desperately needed. Zika virus (ZIKV) infects and kills aggressive cancer cells with stem-like properties (CSCs) from both paediatric and adult brain tumours. Whilst T cell recruitment into ZIKV-infected brain tumours is becoming well documented, the specific mechanisms through which they are activated are poorly understood. We address this by employing a combined LC-MS/MS global proteome and immunopeptidome approach to describe, for the first time, human leukocyte antigen (HLA) presentation of ZIKV peptides on the surface of infected brain tumour cells. We first show that HLA class I (HLA-I) antigen processing & presentation is the most highly enriched immune response pathway in the global proteome of aggressive paediatric USP7-ATRT brain tumour cells following ZIKV infection. We identify USP7-ATRT cells as a good immunopeptidome model due to their homozygous of the globally most common HLA-A allotype (A*02:01). We predict the majority of the 19 ZIKV peptides that we identify here to strongly bind and be presented by HLA-A*02:01. We show that immunopeptide presentation corresponds with cellular ZIKV protein abundance, with ten peptides arising from the most abundant viral protein; non-structural protein 3 (NS3). Specifically, we show the ZIKV NS3 helicase domain to be a rich source of peptides. Finally, we verify that the identified ZIKV peptides do not mimic predicted peptides of the human proteome. The ZIKV peptides we identify here are potential candidates for developing novel epitope-specific brain tumour immunotherapies, and our findings provide potential insight into the efficacious cytotoxic T cell response that oncolytic ZIKV virotherapy can induce against brain tumours. Author Summary Viruses can attack cancer through two mechanisms: 1) infecting and killing the cancer cell and 2) activating the immune system against the tumour. Zika virus (ZIKV) uses both mechanisms to fight brain cancer. Here, we employ a powerful proteomic technique to identify fragments of viral proteins (peptides) presented by cell surface receptors on brain cancer cells infected with ZIKV. In the human body, immune system cells such as T cells recognise and become activated in response to these viral peptides and subsequently attack the infected patient tumour. We identify 19 ZIKV peptides, three of which have been shown previously to elicit T cell responses, four identified elsewhere, and twelve are novel. Our work helps delineate a component of how ZIKV acts as an immunotherapy, the T cell-specific immune response that the virus raises to promote clearance of brain tumours. The significance of our study is that the ZIKV peptides we identify may lead to the development of a novel brain tumour immunotherapy.

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused an epidemic in 2015-2016 in the Americas and raised serious global health concerns due to its association with congenital brain developmental defects in infected pregnancies. Upon infection, ZIKV assembles virus particles in a virus-generated toroidal compartment next to the nucleus called the replication factory, or viroplasm, which forms by remodeling the host cell endoplasmic reticulum (ER). How the viral proteins control viroplasm assembly remains unknown. Here we show that the ZIKV non-structural protein 3 (NS3) is sufficient to drive the assembly of a viroplasm-like structure (VLS) in human cells. NS3 encodes a dual-function protease and RNA helicase. The VLS is similar to the ZIKV viroplasm in its assembly near centrosomes at the nuclear periphery, its deformation of the nuclear membrane, its recruitment of ER, Golgi, and dsRNA, and its association with microtubules at its surface. While sufficient to generate a VLS, NS3 is less efficient in several aspects compared to viroplasm formation upon ZIKV infection. We further show that the helicase domain and not the protease domain is required for optimal VLS assembly and dsRNA recruitment. Overall, this work advances our understanding of the mechanism of viroplasm assembly by ZIKV and likely will extend to other flaviviruses. Importance The Zika virus replicates its genome and assembles virus particles in the cytoplasm within the replication organelle, a large virus-induced compartment also called the viroplasm. It does this in part by remodeling the endoplasmic reticulum. However, how the virus directs the host cell to assemble the viroplasm is mostly unknown. This study shows that Zika virus non-structural protein 3 (NS3) is sufficient to assemble a viroplasm-like structures, and indicates that NS3 has a central role in assembling the viroplasm. Understanding how the virus assembles the viroplasm compartment and NS3’s role in it should significantly advance our understanding of the cellular mechanisms of virus infection. This study aims to gain more understanding of the Zika virus and its viroplasm along with the molecular mechanisms for viroplasm assembly which might be shared by other viruses.

Background Dengue virus (DENV) and Zika virus (ZIKV) are primarily transmitted by Aedes mosquitoes. As most studies on vector competence have focused on Aedes aegypti and Aedes albopictus while neglecting other Aedes species, it is possible that the transmission risks might be underestimated. it is necessary to examine additional species that could potentially serve as competent vectors. This is particularly important considering the potential expansion of their geographical range due to climate change or species-specific vector reduction interventions. Methodology/Principal Findings In this study, we examined the infection kinetics and transmission potential of Aedes scutellaris from Thailand, comparing to Ae. aegypti and Ae. albopictus . Our findings demonstrated that Ae. scutellaris and Ae. albopictus had lower rates of midgut infection compared to Ae. aegypti due to smaller blood meal sizes during feeding. However, once the infection has established Ae. scutellaris exhibited efficient replication of ZIKV and DENV1-4 in the midguts, secondary organs, and salivary glands. Notably, Ae. scutellaris had a low salivary gland escape barrier, with comparable transmissibility as Ae. aegypti when inoculated with the same viral load. Conclusion This study highlights the potential of Ae. scutellaris as a vector for DENV and ZIKV and emphasizes the importance of considering neglected mosquito species in arbovirus transmission and surveillance efforts. Author Summary Dengue and Zika are viral infections caused by arthropod-borne flaviviruses, and spread primarily through the bite of infected Aedes mosquitoes. Most research on DENV and ZIKV transmission has primarily focused on Aedes aegypti and Aedes albopictus while other Aedes species are overlooked, thus the epidemiology of the transmission might be underestimated. With climate change together with species-specific mosquito population reduction interventions these neglected Aedes species could become increasingly important in sustaining virus transmission. In this study, we examined Aedes scutellaris , a mosquito species that co-habitats with Ae. aegypti and Ae. albopictus , to assess its ability to transmit DENV and ZIKV using a combination of blood feeding and intrathoracic injection methods. Our findings show that although Ae. scutellaris had lower initial infection rates due to smaller blood meals, DENV and ZIKV were able to replicate and transmit at levels comparable to Ae. aegypti when exposed to similar virus loads. This highlights the need to study a broader range of species to improve virus control and outbreak prevention strategies.

Flavivirus assembly at the endoplasmic reticulum is driven by the structural proteins envelope (E) and premembrane (prM). Here, contrary to the established paradigm for flavivirus assembly, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM nor proteolytic activation. The expression of E preceded by a truncated version of prM (M-E) was sufficient for the formation of non-infectious Zika virus subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher quality and broadly applicable approach to flavivirus vaccination.

ABSTRACT N-linked glycosylation of flavivirus envelope proteins is widely viewed as a requirement for optimal folding, processing and/or transit of envelope proteins, and the assembling virons, through the endoplasmic reticulum (ER) and Golgi. Herein we show that serial passage of ZIKV Natal in Rag1 -/- mice generated two unique envelope glycan-deficient mutants, ZIKV-V153D and ZIKV-N154D, that, surprisingly, produced titers ∼1 to 2.6 logs higher than the glycosylated parental ZIKV Natal in Vero E6 cells and human brain organoids. RNA-Seq of infected organoids suggested that this increased replication fitness was associated with upregulation of the unfolded protein response (UPR). Cell death, cellular viral RNA and viral protein levels were not significantly affected, arguing that these glycan mutants enjoyed faster ER/Golgi folding, processing, assembly, transit, and virion egress, assisted by an upregulated UPR. Thus, ZIKV envelope N-linked glycosylation is not essential for promoting envelope folding, assembly and transit through the ER/Golgi, as aspartic acid (D) substitutions in the glycosylation motif achieve this with significantly greater efficiency. V153D and N154D mutants have not been employed in flavivirus envelope glycosylation studies. Instead, mutants such as N154A have been used, which may impart unfavorable properties that have a greater impact than the loss of the glycan. ZIKV-V153D and -N154D may avoid this by preserving the surface negative charge provided by the glycan moiety in the parental ZIKV Natal . In Ifnar -/- mice ZIKV-V153D and -N154D showed faster viremia onsets, but reduced viremic periods, than the parental ZIKV Natal , consistent with the contention that these glycans have evolved to delay neutralizing antibody activity. IMPORTANCE Studies seeking to understand the role(s) of N-linked glycosylation of flavivirus envelope proteins often introduce amino acid substitutions that disrupt the glycosylation motif, which in ZIKV has the sequence 153 VNDT 156 . Unfortunately, such substitutions, for instance N154A, may themselves impart unfavorable properties on envelope that have a greater impact than the loss of the glycan moiety. Herein we describe two unique glycosylation motif mutants, ZIKV-V153D and -N154D that were positively selected during passage of ZIKV Natal in Rag1 -/- mice. These N 154 glycan-deficient viruses produced viral titers up to ∼400 fold higher than the parental ZIKV Natal in Vero cells and in human brain organoids. Such glycans are thus clearly not a requirement for optimal folding and trafficking of ZIKV envelope through the endoplasmic reticulum/Golgi. These results provide new insights into the molecular mechanisms underpinning viral fitness in vitro and in vivo , and also have implications for virus-like-particle vaccine design and production.

In early 2024, explosive outbreaks of Oropouche virus (OROV) linked to a novel viral lineage were documented in the Brazilian Amazon. Here, we report the introduction of this emerging orthobunyavirus into Colombia and its co-circulation with another OROV lineage. This investigation highlights the complex arbovirus dynamics in South America.

Mosquito borne flaviviruses, including dengue (DENV) and Zika (ZIKV) viruses, have caused global epidemics in areas with high HIV prevalence due to the expanded geographic range of arthropod vectors. Despite the occurrence of large flavivirus outbreaks in countries with high HIV prevalence, there is little knowledge regarding the effects of flavivirus infection in people living with HIV (PLWH). Here, we use a pigtail macaque model of HIV/AIDS to investigate the impact of simian immunodeficiency virus (SIV)-induced immunosuppression on ZIKV replication and pathogenesis. Early acute SIV infection induced expansion of peripheral ZIKV cellular targets and increased innate immune activation and peripheral blood mononuclear cells (PBMC) from SIV infected macaques were less permissive to ZIKV infection in vitro . In SIV-ZIKV co-infected animals, we found increased persistence of ZIKV in the periphery and tissues corresponding to alterations in innate cellular (monocytes, neutrophils) recruitment to the blood and tissues, decreased anti-ZIKV immunity, and chronic peripheral inflammatory and innate immune gene expression. Collectively, these findings suggest that untreated SIV infection may impair cellular innate responses and create an environment of chronic immune activation that promotes prolonged ZIKV viremia and persistence in the gastrointestinal tract. These results suggest that PLWH or other immunocompromised individuals could be at a higher risk for chronic ZIKV replication, which in turn could increase the timeframe of ZIKV transmission. Thus, PLWH are important populations to target during the deployment of vaccine and treatment strategies against ZIKV. Author Summary Flaviviruses, including Zika virus (ZIKV), cause global epidemics in areas with high HIV prevalence. Yet questions remain as to whether ZIKV disease is altered during an immunocompromised state and the potential immune mechanisms contributing to enhanced disease. This is essential to our understanding of ZIKV disease in people living with HIV (PLWH). Here, we use a non-human primate (NHP) model of HIV/AIDS to investigate the impact of immune suppression on ZIKV replication and pathogenesis. The use of the NHP model was critical for the assessment of longitudinal specimens across tissues that are active sites of flavivirus replication and host immune responses. This study broadly demonstrates that ZIKV pathogenesis is altered and more persistent in states of immunosuppression. Collectively, this study suggests that in PLWH and immunocompromised individuals, other arboviruses, including dengue and West Nile viruses, could similarly alter pathogenesis and/or viral peristance in tissues. Furthermore, this study highlights the need to prioritize immunocompromised individuals in the design and rollout of vaccines against arboviral diseases.

Abstract Background Urban arboviruses pose a significant global burden, particularly in tropical regions like Brazil. São Sebastião, a lower-middle-class urban area just 26 km from the Brazilian capital, is a hyperendemic area for dengue cases. However, asymptomatic cases may obscure the actual extent of the disease. In this study, we measured the seroprevalence of dengue, Zika virus, and chikungunya, and compared these findings with surveillance data. Methods A cross-sectional study was conducted involving 1,535 households. ELISA serological tests were performed to detect IgM and IgG antibodies against dengue, Zika virus, and chikungunya. History of previous exposure to arbovirus, data on age, gender, and education level were collected through a questionnaire. Participants testing positive for IgM and/or IgG were classified as positive. Statistical analyses included tests for normality, associations, mean comparisons, and correlations. Positive serological results were compared with cases captured by local epidemiological surveillance. Results The study included 1,405 individuals, divided into two groups related to pre-pandemic and pandemic COVID-19 phases. Among participants, 0,7% to 28,8% self-reported history of dengue, Zika, or chikungunya. However, the estimated overall seroprevalence was 64.3% (95% CI: 61.8–66.7) for dengue virus, 51.4% (95% CI: 48.8–53.9) for Zika virus, and 5.4% (95% CI: 4.4–6.7) for chikungunya virus. Multiple arboviruses were noted at 4.0% (95% CI: 3.1–5.1). Advancing age and lower education were associated with higher exposure to arboviruses (p < 0.05). The number of urban arboviral cases was 84 times higher than reported. Conclusions The underreporting of arbovirus cases in the evaluated area reveals a substantial deficiency in epidemiological surveillance. Addressing these gaps is crucial for effective resource allocation and implementation of public health interventions.

ABSTRACT Plaque assay is the gold standard for the quantification of viable cytopathic viruses like Zika virus (ZIKV). Some strains of ZIKV produce plaques that are very difficult to accurately visualize and count on the commonly used Vero cell line. From data generated in our lab, we became curious if Vero/TMPRSS2 cells may be a better alternative, therefore we compared the plaque forming units (PFU) of two strains of ZIKV on Vero/TMPRSS2 cells to those produced by Vero cells. We also compared the virus stock titer generated on Vero/TMPRSS2 cells to that generated by the Vero cell line. Although Vero cells generated higher quantity of ZIKV stocks, Vero/TMPRSS2 cells produced plaques with significantly improved morphology and visibility and may therefore be a better alternative to use for performing plaque assays for strains of ZIKV that are more difficult to titer on regular Vero cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused a global pandemic in 2016-2017 with continued ongoing transmission at low levels in several countries. In the absence of an approved ZIKV vaccine, neutralizing monoclonal antibodies (mAbs) provide an option for the prevention and treatment of ZIKV infection. Previous studies identified a potent neutralizing human mAb ZIKV-117 that reduced fetal infection and death in mice following ZIKV challenge. In this study, we report exquisite potency of ZIKV-117 in a titration study in rhesus macaques to protect against ZIKV challenge. We show complete protection at a dose of 0.016 mg/kg ZIKV-117, which resulted in median serum concentrations of 0.13 µg/mL. The high potency of this mAb supports its potential clinical development as a novel biotherapeutic intervention for ZIKV. Importance In this study, we report the potency of the ZIKV-specific neutralizing antibody ZIKV-117 against ZIKV challenge in a titration study rhesus macaques. This high potency supports the further development of this mAb for ZIKV.

Background: ; Cell-released extracellular vesicles (EVs) acting as 'metabolically and proteolytically active machines,' show potential in metabolomic and proteomic analysis of serum EVs. Despite diverse challenges, post-isolation omics characterization EVs offers crucial insights for effective analysis; (2) Methods: The research, involved children with Congenital Zika Syndrome, utilizing mass spectrometry for proteomics and GC-MS for metabolite identification. Vesicles were isolated using Izon qEV columns, quantified, and characterized by NTA and TEM. Data analysis employed Cytoescape/String and MetaboAnalyst, revealing variations in metabolic and proteomic profiles among groups through PCA and volcano plots. Proteins and Metabolite set enrichment analysis provided biologically meaningful patterns to enriched metabolites; (3) Results: Using molecular exclusion chromatography, the EVs were characterized, revealing size variations. Protein analysis identified 13 significantly altered proteins, including upregulated (e.g., AOM8Q6 - IGLC7) and downregulated (e.g., Q8TD86 - CALML6) ones. Metabolite analysis indicated involvement in the PI3K-AKT-mTOR pathway and suggested a role in Angiotensin inhibition in CZS+. Upstream of mTOR, Akt is the central signaling molecule in the PI3K pathway and plays critical roles in brain development as well as synaptic plasticity important for Zika Virus. The study provides insights into molecular mechanisms associated with CZS; (4) Conclusions: The study pinpointed valuable possible biomarkers, specifically proteins and metabolites, in Zika virus (ZIKV) infection. It stresses the necessity for broader investigations with advanced techniques to uncover molecular targets, potentially advancing pharmacological strategies.