ABSTRACT Congenital viral infections can have severe consequences for pregnancy and fetal outcomes. Remarkably, the fetal-derived placenta serves as a robust barrier to infection through meticulous regulation by immune effectors and a diverse repertoire of cytokines. Yet, the regulatory roles of many cytokines remain undefined at the maternal-fetal interface. Interleukin 27 (IL-27) is a highly expressed cytokine in the placenta whose functional consequence during congenital infection is unknown. Here, we utilized trophoblast organoids (TO) derived from primary human placentas and a mouse model of congenital viral infection to uncover the functional role of IL-27 signaling during pregnancy. We show that TOs constitutively express IL-27 and its receptor, IL27RA, and demonstrate that IL-27 signaling restricts Zika virus (ZIKV) infection of TOs. Through bulk RNA-sequencing of TOs in the absence and presence of IL-27 signaling, we demonstrate IL-27-mediated upregulation of antiviral genes. Finally, we show that IL-27 signaling is critical within the context of congenital murine ZIKV infection, as IL-27 restricts placental ZIKV burdens and protects against pathologic fetal outcomes early in gestation. These findings collectively demonstrate a novel role for IL-27 in the placenta and establish IL-27 as an innate antiviral defense at the maternal-fetal interface during congenital viral infection.

Epidemiological models of mosquito-borne virus transmission often lack accurate estimates of host-to-vector transmission probability. Here, we estimated this probability for two strains of Zika virus (ZIKV)—one sylvatic and one human-endemic—from two monkey species to Aedes albopictus mosquitoes using experimental infection data. Viral dynamics did not differ between monkey species, although one (cynomolgus macaque) is a native ZIKV host and the other (squirrel monkey) a novel host, but differed between strains, with viremia for the human-endemic strain peaking later and lower than the sylvatic strain. Only the sylvatic strain was transmitted to mosquitoes. In mosquitoes, anatomical barriers influence viral progression to salivary glands, complicating host infectiousness estimation. We quantified the probability of viral dissemination to the legs in Ae. albopictus , which increased with host viral load and was higher after feeding on squirrel monkeys than on cynomolgus macaques. We also found a positive relationship between virus titer in mosquito legs and virus detection in saliva after a 14-day extrinsic incubation period. Combining these factors, we found that squirrel monkeys were on average 1.5 times more infectious to Ae. albopictus than cynomolgus macaques. These estimates will help assess ZIKV’s potential to establish an enzootic, sylvatic cycle in the Americas.

Abstract MDA5 is an innate immune RNA sensor that senses infection with a range of viruses and other pathogens. MDA5’s RNA agonists are not well defined. We used single-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) to study its ligands. Surprisingly, upon infection with SARS-CoV-2 or encephalomyocarditis virus, MDA5 bound overwhelmingly to cellular RNAs. Many binding sites were intronic and proximal to Alu elements and to potentially base-paired structures. Concomitantly, cytoplasmic levels of intron-containing unspliced transcripts increased in infected cells and displayed enrichment of MDA5 iCLIP peaks. Moreover, overexpression of a splicing factor abrogated MDA5 activation. Finally, when depleted of viral sequences, RNA extracted from infected cells still stimulated MDA5. Taken together, MDA5 surveys RNA processing fidelity and detects infections by sensing perturbations of posttranscriptional events such as splicing, establishing a paradigm of innate immune ‘guarding’ for RNA sensors.

Abstract Background : Zika virus (ZIKV) is a mosquito-borne Flavivirus with a strong affinity for the central nervous system (CNS). After infection, ZIKV can cross the blood-brain barrier (BBB) and reach the CNS, causing potential harm to both adult and developing brains. Methods : The current study aims to evaluate how dysregulated circadian rhythms can affect brain infection by ZIKV, as biorhythms regulate essential physiological processes and disrupted circadian clock can contribute to the pathogenesis of multiple disorders. Both ZIKV infection and circadian rhythm alterations have been related to the disruption of the BBB integrity by modulating the expression of the tight junction (TJ) proteins, however, the input of circadian misalignment on ZIKV infection has never been studied in the literature. Results : Infection of brain endothelial cells with ZIKV selectively impacted endothelial permeability and dysregulated the expression of TJ and mitochondrial proteins. Importantly, these effects were potentiated by silencing Bmal1, a critical circadian rhythm gene. These results were then confirmed in vivo in Bmal1 endothelial cell-specific knockout mice, which were infected with ZIKV at 105 PFU (plaque-forming unit) by retro-orbital infusion. ZIKV infection resulted in a marked decrease in claudin-5, occludin, JAM-3, and ZO-1 expression levels in these mice. In addition, ZIKV affected the expression of FIS1 protein levels and the respiratory complexes of II, III, and IV in mice lacking Bmal1 expression in endothelial cells. Conclusions : Findings from this study contribute to a better understanding of the impact of circadian misalignment on the pathology of ZIKV infection in the adult brain.

ABSTRACT Metagenomics is a powerful tool for characterising viruses, with broad applications across diverse disciplines, from understanding the ecology and evolutionary history of viruses to identifying causative agents of emerging outbreaks with unknown aetiology. Additionally, metagenomic data contains valuable information about the amount of virus present within samples. However, we have yet to leverage metagenomics to assess viral load, which is a key epidemiological parameter. To effectively use sequencing outputs to inform transmission, we need to understand the relationship between read depth and viral load across a diverse set of viruses. Here, using target enrichment sequencing, we investigated the detection and recovery of virus genomes by spiking known concentrations of DNA and RNA viruses into wild rodent faecal samples. In total, 15 experimental replicates were sequenced with target enrichment sequencing and compared to shotgun sequencing of the same background samples. Target enriched sequencing recovered all spike-in viruses at every concentration (10 2 , 10 3 , and 10 5 ± 1 log genome copies) and showed a log-linear relationship between spike-in concentration and mean read depth. Background viruses (including Kobuvirus and Cardiovirus ) were recovered consistently across all biological and technical replicates, but genome coverage was variable between virus genera and likely reflected the composition of target enrichment probe panel. Overall, our study highlights the strengths and weaknesses of using commercially available panels to quantify and characterise wildlife viromes, and underscores the importance of probe panel design for accurately interpreting coverage and read depth. To advance the use of metagenomics for understanding virus transmission, further research will be needed to elucidate how sequencing strategy (e.g. library depth, pooling), virome composition, and probe design influence viral read counts and genome coverage.

Abstract Zika, a mosquito-borne flavivirus, has been found in 87 countries and territories. Global outbreaks peaked in 2016. Prenatal infection of Zika virus was found to be associated with microcephaly, arthrogryposis, intracranial calcifications, fetal growth restriction, and fetal demise. The most severely affected children were diagnosed with congenital Zika syndrome, which impacts thousands worldwide. With no approved treatment or preventative measures for Zika, future viral outbreaks have the potential to cause epidemic levels of prenatal brain injury, as seen over the past 70 years. Therefore, there is a great need for a reliable and clinically translational experimental system that mimics the human condition of prenatal Zika infection. To this end, we developed a humanized, immunocompetent mouse model system of virally induced brain injury from prenatal Zika infection, which ranges from mild to severe. Here, we describe the extent to which this system mirrors the human phenotypic spectrum. Using our thorough preclinical system, we find that prenatal Zika infection of mice impacts survival rate, anthropometric measurements, tissue formation, and neurological outcomes, all of which are typical of prenatal infection. Single-cell RNA sequencing of the Zika-infected cerebral cortex reveals severely disrupted transcriptome profiles and suggests that these injuries are a result of a depletion of neural stem cells. Current and future applications include the identification of genetic or environmental modifiers of brain injury, molecular or mechanistic studies of pathogenesis, and preclinical evaluation of future therapies.

Hybridization capture approaches can significantly increase the sensitivity of metagenomics. However, their implementation with Oxford Nanopore Technologies (ONT) sequencing has been limited due to the lack of dedicated workflows and the need for an extra ligation-based library preparation step. We developed a universal four-primer PCR approach that allows the implementation of hybridization capture workflows with ONT without additional ligation-based steps. Our method allowed the fast conversion of a targeted metagenomics workflow, increasing ONT sensitivity by 10 to 100-fold compared to untargeted approaches and providing both rapid detection and whole-genome sequences where the pathogen was abundant and identification where viral loads were low.

Abstract Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged as a global health threat. The emergence of ZIKV has increased the incidence of neonates born with microcephaly or other neurological syndromes. The majority of ZIKV infections are mild or asymptomatic; however, clinical diagnosis is inaccurate. Moreover, Dengue virus cross-reacts with Zika antibodies, which creates problems for the serological diagnosis of ZIKV infections. Zika serological assays are often performed dismally in dengue-endemic areas because of this phenomenon. In this study, we established a Zika/Dengue ELISA Test to improve the differential diagnosis between Zika and Dengue samples. Sixty Zika-positive samples and 120 controls (20 Primary Dengue samples, 80 Secondary Dengue samples, and 20 healthy serum samples were tested using a ZNS1 and DNS1 Indirect ELISA and a commercial IgG ELISA Kit. Different Zika antigens (EDIII, MR766 NS1 and SV0127 NS1) were tested and ROC curves were compared. Among the antigens tested, NS1 yielded the best diagnostic potential with an AUC range of 0.84–0.88, compared with an AUC of 0.77–0.82 for EDIII. The Zika/Dengue OD Ratio also exhibited the best sensitivity (Sn) and specificity (Sp) (58.3% and 79.2% respectively) among the other parameters tested (Sn = 26.7%–28.3% and Sp = 59.2%–79.3%). It also performed better than the commercial kit, which yielded Sn and Sp values of only 26.7% and 74.2%, respectively. The Zika/Dengue OD Ratio has diagnostic potential and better performance compared with commercial ELISA test kits for detecting Zika infections. The combination of two simple ELISAs may be applied for ZIKV serosurveys and to monitor ZIKV infection during pregnancy to understand the epidemiology, pathogenesis, and complications of ZIKV in DENV-endemic areas.

Arboviruses, including dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV), pose a significant global health and economic burden, with Aedes aegypti serving as their primary vector. Arbovirus infection in Ae. aegypti progresses sequentially through the midgut (MG), carcass (CA), and salivary glands (SG), with each tissue exhibiting distinct antiviral responses. Here, we investigate tissue-specific antiviral mechanisms, focusing on the small interfering RNA (siRNA) pathway in SGs. Our results reveal that SGs possess weaker antiviral defense and are more susceptible to arboviral infection compared to MGs and CAs. Notably, overexpression of Dicer2 ( Dcr2 ), a key component of the siRNA pathway, in SGs leads to a significant decrease in arboviral replication. Conversely, Dcr2 overexpression in fat bodies, the primary tissue in CAs, only moderately suppresses DENV2 infection and has no notable effect on Mayaro virus (MAYV) infection. Remarkably, the simultaneous overexpression of Dcr2 in both MGs and SGs enhances antiviral activity, effectively blocking the transmission of multiple arboviruses. These findings reveal the tissue-specific dynamics of mosquito antiviral immunity and underscore the potential for targeting SG-specific immunity to disrupt arbovirus transmission, providing a promising approach for controlling mosquito-borne diseases.

A select group of pathogens infect neurons in the brain. Prior dogma held that neurons were “defenseless” against infecting microbes, but many studies suggest that neurons can mount anti-microbial defenses. However, a knowledge gap in understanding how neurons respond in vitro and in vivo to different classes of micro-organisms remains. To address this gap, we compared a transcriptional dataset derived from primary neuron cultures (PNCs) infected with the neurotropic intracellular parasite Toxoplasma gondii with a dataset derived from neurons injected with T. gondii protein in vivo . These curated responses were then compared to the transcriptional responses of PNCs infected with the single stranded RNA viruses West Nile Virus (WNV) or Zika Virus (ZKV). These analyses highlighted a conserved response to infection associated with chemokines ( Cxcl10, Ccl2 ) and cytokines (interferon signaling). However, T. gondii had diminished IFN-α signaling in vitro compared to the viral datasets and was uniquely associated with a decrease in neuron-specific genes ( Snap25 , Slc17a7 , Prkcg ). These data underscore that neurons participate in infection-induced neuroinflammation and illustrate that neurons possess both pathogen-specific and pathogen-conserved responses. Importance Though neurons are commonly the target of pathogens that infect the CNS, few datasets assess the neuronal response to infection. This paucity of data is likely because neurons are perceived to have diminished immune capabilities. However, to understand the role of neurons in neuroinflammation and their immune capabilities, their responses must be investigated. Here we analyzed publicly accessible, neuron-specific datasets to compare neuron responses to a eukaryotic pathogen versus two Orthoflaviviruses. A better understanding of neuron responses to different infections will allow us to develop methods for inhibiting pathways that lead to neuron dysfunction, enhancing those that limit pathogen survival, and mitigating infection-induced damage to the CNS.

Respiratory RNA viral infections significantly impact quality of life and productivity, both in terms of pandemics (e.g., SARS-CoV-2, influenza A viruses) and seasonal infections such as respiratory syncytial virus (RSV). Globally, RSV causes an estimated 33.8 million cases annually in children under five, leading to 2.8–4.3 million hospital admissions and up to 199,000 deaths. Human-relevant drug screening models are key to addressing the urgent need for effective antiviral treatments for RSV, particularly in vulnerable patient groups. Here, we present a donor-derived differentiated primary human nasal epithelial cell, 96-HTS Transwell air-liquid interface (ALI) model designed to investigate the effects of combination antiviral therapies on RSV infection in primary human ciliated airway epithelium. Additionally, we describe novel analytical tools using R ( ciliR ) to screen drug combinations by concurrently measuring efficacy and ciliary beat frequency as a sensitive marker of cell toxicity. Our results demonstrate that the smaller 96-HTS ALI cultures retain comparable epithelial composition and ciliary function to conventional ALI culture formats. These cultures are permissible to infection with an RSV-GFP reporter virus, enabling quantitative comparison and combination treatment across multiple epithelial cultures from the same donor. We anticipate that our disease-relevant system will serve as a foundation for larger-scale experiments aimed at optimizing combination therapy for RSV and other respiratory viruses.

ABSTRACT Flavivirus infections by Dengue and Zika virus impose a significant healthcare threat worldwide. At present no FDA-approved specific antiviral treatment is available, and the safety of a vaccine against Dengue virus is still under debate. Here, we report the identification of the CADA derivative flavitransin (FT), with potent activity against DENV serotype 2 in various cell types. Moreover, FT showed consistent anti-flaviviral activity against all four DENV serotypes, and also against Zika and Yellow fever virus. Viral polyprotein biogenesis was completely abolished by FT treatment of DENV-infected cells. Drug profiling by a-time-of-drug-addition assay revealed a post-entry antiviral effect of FT, in line with its anticipated Sec61 inhibitory effect. Subsequent analysis of the individual viral proteins in transfected HEK293T cells indicated that FT suppresses the expression of the structural proteins (pre-membrane and envelope) only. Furthermore, cell free in vitro protein translation analysis demonstrated a direct inhibitory effect of FT on the co-translational translocation of the DENV polyprotein across the ER membrane. More specifically, FT inhibited the initiation of protein translocation into the ER that relies on the N-terminal transmembrane region of the capsid subunit of the DENV polyprotein, resulting in rerouting of the viral pre-protein to the cytosol for proteasomal degradation. Finally, selection and genotyping of FT-resistant HCT116 cells revealed a unique A70V mutation in the Sec61α subunit that conferred resistance to FT in infected cells. Long-term exposure of DENV to FT demonstrated a high barrier to resistance development. In conclusion, our data demonstrate that FT selectively interferes with the initiation of ER co-translational translocation of the DENV polyprotein and confirm the critical role of this translocation process in the flavivirus replication cycle.

Introduction Measures of maternal fractional blood volume (mFBV) in the placenta holds potential to diagnose placental vasculature deficiencies. However, methods for quantitative mapping of blood volume are challenging to implement for clinical placenta evaluation. As a preliminary step towards human applications, this study assesses the feasibility of blood volume measurements using ferumoxytol enhanced variable flip angle (VFA) T1-mapping in Zika-infected rhesus macaques. Methods Seven pregnant rhesus macaques were imaged longitudinally at up to 3 timepoints across gestation (days 64.5±1.9, 100.8±3.9, and 145.3±1.8), corresponding to first, second, and third pregnancy trimester of the rhesus. Four animals received a Zika virus (ZIKV) injection into the amniotic fluid, while three control rhesus macaques received a saline injection. T1-weighted spoiled gradient echo sequences at four flip angles (2°, 6°, 10°, 14°) were used for quantitative mFBV assessment derived from pre- and post-contrast T1 mapping using ferumoxytol. Image quality assessment and segmentation assessment was performed on the full 3D coverage. Placental histopathology for all animals was analyzed by a professional pathologist with over 15 years of experience. Results All scans were successfully acquired and analyzed with no significant motion artifacts. 3D mFBV maps show regional heterogeneities within slices. FBV and total placental blood volume has an increasing trend with gestation. Discussion This study shows feasibilities to measure mFBV in non-human primates using ferumoxytol enhanced VFA T1-mapping.

Background: From the first isolation of Zika virus (ZIKV) in Uganda in 1947, ZIKV had primarily been associated with sporadic human cases in Africa and Asia until ZIKV emerged as an epidemic in the Americas in 2015. As ZIKV spread into new geographic regions, it now has the potential to interact with many novel potential host species whose susceptibility to the virus has yet to be determined. Bats, with their ability to fly and live in or near human structures, are plausible ZIKV reservoirs. However, their competence as hosts for ZIKV remains unresolved. In this study, we investigate the immune response of Old World and New World bats to ZIKV infection in vitro. Results: We demonstrate that Egyptian Rousette bat (Rousettus aegyptiacus; Old World) cells are susceptible to ZIKV, while we observed little to no ZIKV replication in Jamaican fruit bat (Artibeus jamaicensis) cells. Notably, both the Asian and African ZIKV lineages elicited a strong proinflammatory response in the R. aegyptiacus cell line including upregulation of IL6, CXCL8, and CCL5. These data contrast with the dampened inflammatory response detected in these bats to other viruses. Conclusions: The findings reveal that R06E cells derived from Egyptian Rousette bats exhibit robust proinflammatory and antiviral responses upon Zika virus (ZIKV) infection, characterized by significant upregulation of proinflammatory cytokines. This suggests that while these cells support productive ZIKV replication, they also mount a strong immune response, challenging the notion of these bats as immune-tolerant reservoirs and indicating a more complex interaction with the virus.

Abstract Brain tumours disproportionately affect children and are the largest cause of paediatric cancer-related death. Novel therapies that engage the immune system, such as oncolytic viruses (OVs), hold great promise and are desperately needed. The Zika virus (ZIKV) infects and destroys aggressive cells from multiple paediatric central nervous system (CNS) tumours. Despite this, the molecular mechanisms underpinning this response are largely unknown. We comprehensively investigate the transcriptomic response of paediatric medulloblastoma and atypical teratoid rhabdoid tumour (ATRT) cells to ZIKV infection. We observe conserved TNF signalling and cytokine signalling-related signatures and show that the TNF-alpha signalling pathway is implicated in oncolysis by reducing the viability of ZIKV-infected brain tumour cells. Our findings highlight TNF-alpha as a potential prognostic marker for oncolytic ZIKV (oZIKV) therapy. Complementing our analysis with a 49-plex ELISA, we demonstrate that ZIKV infection induces a clinically relevant and diverse pro-inflammatory brain tumour cell secretome, including TNF-alpha. We assess publicly available scRNA-Seq data to model how ZIKV-induced secretome paracrine and endocrine signalling may orchestrate the anti-tumoural immune response during oZIKV infection of brain tumours. Our findings significantly contribute to understanding the molecular mechanisms governing oZIKV infection and will help pave the way towards oZIKV therapy.

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol . Using ribosome profiling on coxsackievirus CVA-13 ( E. coxsackiepol ), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

Dengue virus (DENV) is the causative agent of dengue fever and exerts a substantial healthcare burden worldwide. Like other flaviviruses, DENV must undergo membrane fusion with the host cell in order to initiate infection. This membrane fusion occurs following acidification during endocytosis and is pH dependent. Here, we interrogate whether the mechanism of DENV fusion contains an off-pathway state, such has been reported previously for two other flaviviruses - Zika virus and West Nile virus. To do this, we utilize single particle lipid mixing measurements of DENV virus-like particles (VLPs) to tethered liposomes, together with computational modeling inspired by chemical kinetics. By observing and then modeling the pH dependence of single VLP fusion kinetics, we provide evidence that the DENV fusion mechanism must contain an off-pathway state. Measuring the proportion of VLPs undergoing hemi-fusion over time, we also demonstrate that the off-pathway state appears to be slowly reversible over tens of minutes, at least for some virions. Additionally, we find that late endosomal anionic lipids do not appear to influence the off-pathway mechanism to any great extent. In conjunction with the prior reports on Zika virus and West Nile virus, this work indicates that an off-pathway fusion state may be a feature of flavivirus fusion more broadly. We also note that the platform and mechanistic model described in this study may be useful in elucidating the mechanism of action of small molecule inhibitors of flavivirus fusion developed by our group and others. Statement of Significance Dengue virus (DENV) causes dengue fever and infects an estimated hundreds of millions of people annually. To date, there are no specific antiviral drugs for DENV and limited vaccination options, highlighting the need to better understand this important pathogen. In this report, we investigate the mechanism of DENV membrane fusion, an early step in the viral infectious cycle, using a mix of experimental techniques and computer simulations. We find strong evidence that the DENV fusion mechanism contains an off-pathway state, in which it can get stalled prior to membrane fusion. Understanding this off-pathway state could be an avenue to develop antiviral strategies against DENV and other related viruses.

Abstract Zika virus (ZIKV) infection can lead to severe congenital outcomes, yet the mechanisms governing its entry into host cells remain understood. ZIKV is a flavivirus known to exploit multiple cellular receptors and cofactors, particularly in neural cells, where infection can result in congenital Zika syndrome (CZS). Here we show that plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor involved in hemostasis, directly interacts with ZIKV particles and critically enhances viral replication in diverse cell types, including human neural progenitor cells and three-dimensional neural organoids. Our findings reveal that PAI-1 may contribute to ZIKV infection through distinct or complementary pathways, underscoring the virus’s versatile entry mechanisms. Inhibition of PAI-1 via tiplaxtinin (TPX) dramatically reduces viral load and impedes infectious particle release, demonstrating a dose-dependent effect that is especially potent in neural models relevant to CZS. These results highlight PAI-1 as an essential mediator of ZIKV pathogenesis and suggest that targeting PAI-1 function could represent a novel therapeutic avenue. Given the risk of future ZIKV outbreaks and the devastating impact of CZS, interventions aimed at PAI-1 may hold promise for reducing the global burden of ZIKV infection.

Heparanase (Hpa) is the only endoglycosidase enzyme in mammalian cells capable of cleaving heparan sulfate. In addition to its well-known functions in the regulation of glycosaminoglycans integrity, accumulating evidence indicates that Hpa plays vital roles in viral infection, while the mechanisms are not yet fully understood, especially in RNA virus infection. In this study, we report that Hpa functions as a restriction factor for Zika virus (ZIKV) infection. Our results demonstrated that Hpa, but not the enzymatic inactive mutant (Hpa-DM), resulted in degradation of the ZIKV envelope (E) protein, which could be rescued by treatment of the proteasome inhibitor (MG132) and the autophagy inhibitor (NH 4 Cl), separately. Additionally, the ubiquitination of ZIKV E did not show an significant change in the presence of Hpa. Overexpression of Hpa, but not Hpa-DM, dramatically decreased ZIKV infection in different cell models, evidenced by the reduction of viral proteins and a compromised production of infectious virions. This was further confirmed by the results in MEF cells, in which knockout Hpa enhanced ZIKV infection, while overexpression of Hpa suppressed the production of virions. In addition, ZIKV was found to downregulate the Hpa expression, which could counteract the inhibitory effects of Hpa. Altogether, our study discovers an unrecognized role of Hpa in virus infection and demonstrates that Hpa serves as a restriction factor for ZIKV infection. Summary Zika virus (ZIKV), primarily transmitted by mosquitoes, can cause a wide range of symptoms, including myalgia, fever, rash, and severe neurological complications such as microcephaly, epilepsy, and Guillain-Barré syndrome. A deep understanding of ZIKV-host cell interactions, particularly the roles of pro- and anti-viral factors, is essential for the development of effective therapeutic strategies, which are currently unavailable. In this study, we uncover that heparanase (Hpa), the only endoglycosidase enzyme in mammalian cells capable of cleaving heparan sulfate (HS), can degrade the ZIKV envelope (E) protein. Hpa exhibits an inhibitory role in ZIKV infection in different cell models. Additionally, we find that ZIKV downregulates the Hpa expression, which could be used by the virus to mitigate the inhibitory effects of Hpa. Taken together, our study demonstrates Hpa as a restriction factor in ZIKV infection and highlights complex interactions between ZIKV infection and HS machinery enzymes.

We report the first identification of Zika virus in Pakistan following genomic and serological analyses of blood samples from 20 patients with febrile illness. In November 2021, an outbreak of dengue-like illness occurred in the metropolitan city of Karachi. Viral genome capture and sequencing of seven patients revealed six cases of dengue virus serotype 2 and two Zika virus infections, including one dengue and Zika virus co-infection. The next year, following severe flooding, 13 suspected dengue patients were screened by real time qRT-PCR and serology, and 92% (12/13) had evidence of current or recent Zika virus infection. Phylogenetic analyses revealed the Zika viruses originated from Brazil. The most recent observed ancestor dates to 2016, suggesting a prior importation event and ongoing circulation. Our results suggest that Zika virus may be circulating and contributing to disease burden during seasonal Dengue outbreak.

Systemic viral infections with neurotropic potential pose significant global health challenges. The Zika virus (ZIKV) is known for its pronounced neurotropism, with recent infectious clusters raising renewed public health concerns. While research has predominantly focused on congenital populations, growing evidence suggests that the mature central nervous system (CNS) is also vulnerable. However, no study has examined the long-term impact of ZIKV infection on the adult human brain. To address this gap, we studied a rare group of adult ZIKV patients presenting with both peripheral (Guillain-Barré Syndrome; GBS) and CNS-related neurological symptoms. We compared these patients at the chronic stage (5 to 12 months post-infection) to healthy controls and to patients with GBS of non-ZIKV etiology (total N =43). Structural and functional measures included cortical thickness, white matter hyperintensities, diffusion metrics, and resting-state functional connectivity. Despite the rarity of both patient populations, power analyses indicated that our sample size could detect large group differences—effect sizes deemed reasonable given the severity of neurological symptoms in the ZIKV group. Nonetheless, our multimodal analyses yielded null results, with Bayesian statistics (where applicable) providing evidence for a lack of effects. The null findings suggest that chronic ZIKV infection in adults is not associated with brain changes of large magnitude. Importantly, this study offers detailed clinical characterization of a heavily understudied group. In light of recent ZIKV outbreaks, this characterization underscores the need to monitor, study, and provide longitudinal care to adult survivors of severe ZIKV infections.

ABSTRACT RNA viruses, including SARS-CoV-2, Influenza A Virus (IAV), Zika Virus, and Dengue Virus (DENV) pose serious global health challenges by manipulating host cellular mechanisms. SARS-COV-2, in particular exploits host translational machinery to enhance replication and evade immune response. Here, we investigated how SARS-CoV-2 circumvents host immune defenses through RNA - host protein interactions. By integrating multiple datasets, ClusterProfiler, KEGG, Reactome, WikiPathways, and Gene Ontology, we performed functional enrichment analyses on host protein interactions with SARS-CoV-2 RNA. Our results identified key pathways involved in viral replication, translation regulation, and immune evasion. Comparing SARS-CoV-2 interactomes from IAV, Zika, and DENV, we uncovered a subset of 275 common host proteins serving as promising targets for broad-spectrum antiviral strategies. Network analysis highlighted critical translation factors (EEF1A1, EIF4A1, EIF3H) and RNA-binding proteins (NCL, ILF3) as key nodes in viral replication. These findings provide insights into RNA virus pathogenesis and support the development of targeted therapeutics.

Abstract Mosquitoes serve as the primary vectors for several life-threatening pathogens, including malaria, dengue fever, and Zika virus. To effectively control the transmission of these diseases, a variety of integrated vector management strategies are currently employed to reduce mosquito population densities. This paper focuses on the sterile insect technique (SIT) and incompatible insect technique (IIT), which involves the release of sterile male mosquitoes to suppress wild mosquito populations, and delves into the impact of different release strategies on mosquito population dynamics. We established a switched mosquito population suppression model with time delay. We first investigate the existence and stability of the switching ordinary differential equation model, and derive some criteria for determining whether T-periodic solutions are stable or unstable. By taking the initial function as a solution to the delay-free model, we obtain sufficient conditions for existence of a unique or exactly two periodic solutions. Furthermore, we gain necessary and sufficient conditions for nonexis-tence of periodic solutions. Finally, the numerical results have been shown to further highlighted the effectiveness of our proposed model.

Abstract Flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), constitute a significant public health concern with billions of people at risk of infection. Climate change and the expanding geographical distribution of mosquito vectors transmitting flaviviruses have increased their potential to cause large-scale disease outbreaks. The frequency and severity of disease outbreaks highlights the urgent need for a broad-spectrum antiviral agent targeting flaviviruses. In this work, we conducted a comprehensive morphological profiling of approximately 200,000 small molecules through a fluorescence-based high-content imaging platform, which led to the identification of a singular small molecule exhibiting broad-spectrum activity against flaviviruses. Subsequent hit deconvolution against DENV serotype 2 (DENV-2) revealed NS2A as a novel therapeutic target and suggested a mechanism whereby the identified small molecule inhibits the interaction between NS2A and the prM protein, revealing a previously uncharacterized antiviral mechanism of action.

The four dengue virus serotypes (DENV1-4) and the related Zika flavivirus (ZIKV) are major public health concerns worldwide. Primary immunity against ZIKV increases the risk of a subsequent severe DENV2 infection, presenting a significant challenge for developing safe and effective ZIKV vaccines. However, the mechanisms driving this phenomenon remain unclear. Leveraging our long-standing Pediatric Dengue Cohort Study in Nicaragua, we show that serum anti-NS1 IgA antibodies elicited after a primary ZIKV infection drive neutrophil activation and correlate with increased risk of subsequent severe DENV2 disease. Depletion experiments combined with e x vivo functional NETosis assays confirmed that anti-NS1 IgA antibodies drive neutrophil activation in dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Moreover, increased neutrophil degranulation in paired serum samples obtained during the acute DENV2 infection from the same individuals correlated with IgA binding to DENV2 NS1 and preceded the development of vascular leakage. This finding was corroborated in an orthogonal hospital-based study. Thus, serum anti-NS1 IgA enhances neutrophil activation in severe dengue, with implications for prognostics, therapeutics, and vaccines.