The Zika virus (ZIKV) epidemic elicited a rapid commitment to the development of animal models for ZIKV research. Non-human primates (NHPs) and mice have made significant contributions to this research, but NHPs are expensive, have a long gestation period, and are available only in small numbers; and non-genetically modified mice are resistant to infection. To address these deficiencies, we have established the laboratory opossum, Monodelphis domestica, as a small animal model that complements the mouse and monkey models. We developed and validated an indirect ELISA for measuring antibodies to ZIKV in opossums, as well as an immunohistochemistry (IHC) method to detect ZIKV NS1 protein in tissue samples and a PCR method to detect ZIKV RNA in tissue samples. Opossum pups inoculated intracerebrally as embryos, juveniles inoculated by several routes, and mothers that cannibalized inoculated pups, became persistently infected with ZIKV. The virus spread to multiple organs and persisted up to 38 weeks (the latest endpoint of the experiments). A robust humoral immune response was mounted, and high titers of antibodies also persisted for 38 weeks. The results establish M. domestica as natural, non-genetically modified animal model in which ZIKV persists long term after experimental infection, and as a unique animal model for research on ZIKV infection, persistence of infection, and biological responses to infection.

Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast. However, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity due to 5' sgRNA extension as key contributors to low retron editor efficiency in human cells. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA (xrRNA) pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to minimize 5' sgRNA extension. This strategy increased steady-state ncRNA levels and rescued sgRNA activity, leading to increased templated repair. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells and presents an enhanced Eco1 retron editor capable of precise genome editing in human cells from a single integrated lentivirus and, in the context of the nCas9 H840A nickase, without creating double-strand breaks.

ABSTRACT Glioblastoma multiforme (GBM) is an aggressive and lethal form of brain cancer with few effective treatments. In this context, Zika virus has emerged as a promising therapeutic agent due to its ability to selectively infect and kill GBM cells. To elucidate these mechanisms and expand the landscape of oncolytic virotherapy, we pursued a transcriptomic meta-analysis comparing the molecular signatures of Zika infection in GBM and neuroblastoma (NBM). Over-representation analysis of dysregulated coding genes showed significant enrichment of tumor necrosis factor (TNF), NF-κB, and p53 signaling pathways. A refined list of long non-coding RNAs consistently dysregulated in Zika-infected GBMs was also developed. Functional review of these candidates revealed their potential regulatory role in Zika-mediated oncolysis. We performed validation of the less-researched targets in adult and pediatric GBM cell lines and found significant differential regulation, as predicted. Altogether, our results provide novel insights into the molecular mechanisms underlying the effect of Zika on GBM. We highlight potential therapeutic targets that could be further interrogated to improve the efficacy of tumor cell death and the utility of Zika as an adjuvant virotherapy for GBM and other related cancers.

ABSTRACT Encephalomyocarditis virus (EMCV) has for decades served as an important model RNA virus. Although most of the EMCV proteins are obtained via proteolytic cleavage of a long polyprotein, 2B* is expressed from a short overlapping open reading frame via an unusual protein-stimulated temporally dependent ribosomal frameshifting mechanism. The function of 2B* has not yet been characterised, though mutant viruses that are unable to express 2B* have a small plaque phenotype. Here we show that 2B* binds all seven members of the 14-3-3 protein family during virus infection. Binding is dependent on the 2B* C-terminal sequence RRNSS. IFN-β and IL-6 signalling are impeded following overexpression of 2B* but not a truncated version lacking the RRNSS residues, thus suggesting a 14-3-3-dependent role for 2B* in inhibiting MAVS signalling. We also find that this function is distinct from the effect of 2B* on plaque size, as a virus in which 2B* was similarly truncated exhibited near-wildtype plaque size, thus indicating that 2B* also harbours additional functions. This work provides the first identification of a role of 2B* in innate immune antagonism and expands our knowledge of the protein complement of this important model virus. IMPORTANCE Encephalomyocarditis virus (EMCV) infects a range of species, causing economically important reproductive disorders in pigs and encephalitis and myocarditis in rodents. Due to its wide host range, it is an important model pathogen for investigating virus-host interactions. EMCV expresses an accessory protein, 2B*, from an overlapping open reading frame via an unusual ribosomal frameshifting mechanism. Although the frameshifting mechanism has been established, the function of the 2B* protein had not previously been explored. Here, we determined the host proteins to which 2B* binds and found that it specifically binds to the entire 14-3-3 protein family which, among other roles, contribute to the innate immune response to viral infection in mammalian cells. This interaction requires a specific stretch of amino acids at the end of 2B*. By interacting with the 14-3-3 proteins, 2B* blocks immune response activation. Thus, 2B* is a novel antagonist of innate immunity.

In RNA interference (RNAi), long double-stranded RNA (dsRNA) is cleaved by Dicer endonuclease into small RNA interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates adopted sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified mouse Dicer locus to express a truncated variant (Dicer ΔHEL1 ) known to stimulate RNAi. Next, we analyzed how Dicer ΔHEL1/wt mice respond to four RNA viruses: Coxsackievirus B3 (CVB3) and encephalomyocarditis virus (ECMV) from Picornaviridae ; tick-borne encephalitis virus (TBEV) from Flaviviridae ; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae . Increased Dicer activity in Dicer ΔHEL1/wt mice did not elicit any antiviral effect. supporting insignificant antiviral function of endogenous mammalian RNAi in vivo . However, we also report that sufficiently high expression of Dicer ΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo .

Abstract Maturing flaviviruses undergo pH-dependent conformational changes, transitioning from trimeric pre-membrane protein prM and envelope protein E (prM-E) complexes to dimeric E with cleaved prM. However, little is known about the intermediate maturation stages and whether immature virions contribute to infection. Here we demonstrated that immature prM-containing particles of tick-borne flaviviruses—tick-borne encephalitis virus (TBEV), Langat virus, and louping ill virus—exhibited infectivity in mammalian cells under physiological conditions. This finding challenges the long-standing assumption that prM-containing flavivirus particles are non-infectious, as is the case for immature mosquito-borne flaviviruses, including Usutu virus, Zika virus, and West Nile virus (WNV). In a mouse model, prM-TBEV exhibited high pathogenicity, contrasting with the reduced pathogenicity of prM-WNV. Structural modelling, functional validation, and site-directed mutagenesis revealed interactions within the prM-E complex that influence pH sensitivity, furin cleavage site accessibility, and virus infectivity. This study, therefore, elucidates the distinctive dynamics of infectivity of tick- and mosquito-borne flaviviruses.

Summary Mosquito-borne flaviviruses have emerged as global health threats due to their rapid spread and high disease burden. The initial site of virus replication is the mosquito biting site in the host skin, yet the role of host skin bacteria remains unclear. Here, we observed accelerated progression of Zika virus (ZIKV)-induced disease in mice with depleted skin bacteria by topical antibiotic treatment prior to ZIKV infection. Increased viral loads in the blood, draining lymph node, spleen and brain of antibiotic-treated mice suggested faster ZIKV dissemination. Flow cytometry revealed increased frequencies of T cells in the lymph nodes, along with enhanced activation of T cells in the brain upon antibiotic treatment. The antibiotic-induced phenotype was rescued by restoring the skin bacteria prior to infection. Our findings demonstrate an increased susceptibility to ZIKV infection in mice with depleted skin microbiomes, suggesting a potential role of skin bacteria in modulating the infection outcome.

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

Like other positive-sense RNA viruses, SARS-CoV-2 manipulates host lipid metabolism to facilitate its replication by enhancing lipogenesis and lipid droplet formation. In doing so, SARS-CoV-2 infection perturbs bioactive lipid levels associated with the inflammatory response. One of these, Palmitoylethanolamide (PEA) is suppressed during SARS-CoV-2 infection since it activates the Peroxisome Proliferator-Activated Receptor-α (PPAR-α), a transcription factor that suppresses the nuclear factor-B (NF-κB), which is mandatory to sustain SARS-CoV-2 replication. PEA levels are regulated by N-acylethanolamine acid amidase (NAAA), a lysosomal enzyme responsible for catalysing the breakdown of PEA. We hypothesized that NAAA inhibition might interfere with SARS-CoV-2 replication since it will lead PEA to accumulate, activating PPAR-α and, consequently, suppressing NF-κB. Our results reveal that genetic or chemical ablation of NAAA significantly suppresses SARS-CoV-2 replication by three log 10 in human-derived precision-cut lung slices. Therefore, we investigated whether inhibiting NAAA could influence NF-κB activation through the activation of PPAR-α. We observed PPAR-α increased expression in NAAA-/-cells, while PPAR-α expression remained low in infected parental cells. As expected, the elevated PPAR-α expression correlated with a parallel reduction in NF-κB activation when NAAA is ablated. These findings underscore NAAA as an essential host factor for SARS-CoV-2 replication and propose a potential mechanism of action rooted in the attenuation of NF-κB activation during viral replication. Author summary Over the past three years, COVID-19 has claimed nearly 7 million lives worldwide, prompting extensive efforts to find effective treatments. While RNA-based vaccines have been developed rapidly, they alone have not completely halted the spread of the virus, making the search for antiviral therapies crucial. One promising approach targets the anti-inflammatory lipid PEA, which has shown some success in COVID-19 clinical trials. PEA is quickly degraded by the enzyme NAAA. Researchers have found that inhibiting NAAA can enhance and prolong PEA anti-inflammatory effects. NAAA inhibitors have already shown effectiveness in reducing chronic pain and lung inflammation in animal models and have also been effective against Zika virus replication. Our research focused on testing the NAAA inhibitor ARN726 against SARS-CoV-2. In human lung cells and lung tissue samples, ARN726 significantly reduced SARS-CoV-2 replication and inflammation. We discovered that this inhibition suppresses the NF-κB pathway, which the virus uses to fuel its replication and sustain Cytokine storm. Overall, our findings suggest that NAAA inhibitors like ARN726 could be repurposed to combat COVID-19 and potentially other coronaviruses, offering a novel and effective antiviral strategy.

Abstract Although vaccines and treatments have strengthened our ability to combat the COVID-19 pandemic, new variants of the SARS-CoV-2 continue to emerge in human populations. Because the evolution of SARS-CoV-2 is driven by mutation, a better understanding of its mutation rate and spectrum could improve our ability to forecast the trajectory of the pandemic. Here, we used circular RNA consensus sequencing (CirSeq) to determine the mutation rate of six SARS-CoV-2 variants and performed a short-term evolution experiment to determine the impact of these mutations on viral fitness. Our analyses indicate that the SARS-CoV-2 genome mutates at a rate of ~3 ´10 -6 /base per round of infection and that the spectrum is dominated by C®U transitions. Moreover, we discovered that the mutation rate is significantly reduced in regions that form base-pairing interactions and that mutations that affect these secondary structures are especially harmful to viral fitness. These observations provide new insight into the parameters that guide viral evolution and highlight fundamental weaknesses of the virus that may be exploited for therapeutic purposes.

ABSTRACT Arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control the spread of arboviruses focus on harnessing the antiviral immune system of their primary vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown. We conducted a phylogenetic analysis of the Vago gene family in Diptera, which led us to focus on a Vago -like gene that we named VLG-1 . Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti , which revealed a broad impact of VLG-1 on the mosquito transcriptome, affecting several biological processes potentially related to viral replication, including the oxidative stress response. Surprisingly, experimental viral challenge of the VLG-1 mutant line indicated a modest proviral role for this gene during DENV and ZIKV infections in vivo . In the absence of VLG-1 , virus dissemination throughout the mosquito’s body was slightly impaired, albeit not altering virus transmission rates. Our results challenge the conventional understanding of Vago -like genes as antiviral factors and underscore the need for further in vivo research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions.

Background: The Zika virus (ZIKV) infection has emerged as a global health threat. The causal reasoning is that Zika infection is linked to the development of microcephaly in newborns and Guillain-Barré syndrome in adults. With no clinically approved antiviral treatment for ZIKV, the need for the development of potential inhibitors against the virus is essential. In this study, we aimed to screen phytochemicals from papaya ( Carica papaya L. ) against NS5 protein domains of ZIKV. Methods Approximately 193 phytochemicals from an online database (IMPACT) were subjected to molecular docking using AutoDock Vina against the NS5-MTase protein domain (5WXB) and -RdRp domain (5U04). Results Our results showed that β-sitosterol, carpaine, violaxanthin, pseudocarpaine, Δ7-avenasterols, Rutin, and cis-β-carotene had the highest binding affinity to both protein domains, with β-sitosterol having the most favorable binding energy. Furthermore, ADMET analysis revealed that selected compounds had good pharmacokinetic properties and were nontoxic. Conclusions Our findings suggest that papaya-derived phytochemicals could be potential candidates for developing antiviral drugs against ZIKV. However, further experimental studies using cell lines and in vivo models are needed to validate their efficacy and safety.

ABSTRACT The development of mammals is a highly complex process, characterized by the necessity for precise concentration- and time-dependent signaling for correct pattern formation and morphogenesis. Despite considerable technological advancements and knowledge gathered, numerous aspects of mammalian development remain elusive. When examining the entire organism, it becomes challenging to disentangle the effects of individual pathways or the mechanism by which external stimuli guide the interference of surrounding tissues and factors. In addressing this complexity, three-dimensional (3D) in vitro models such as organoids have emerged as valuable tools. Organoids, derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), exhibit tissue-like features that closely resemble their in vivo counterparts in terms of expression patterns and functionality. Importantly, they offer accessibility for manipulation and extensive biological studies within a controlled experimental setting. Despite originating from pluripotent cultures, organoid systems often exhibit heterogeneity and substantial variability, limiting their utility when studying complex and intricate biological questions. Therefore, there is a pressing need for detailed protocols aimed at harmonizing procedures that result in high-quality reproducible data, reduction of materials used and which importantly permit the investigation of convoluted phenomena. In this context, we present an optimized protocol for the cultivation of neural tube organoids (NTOs) in vitro . By producing stable culture conditions and offering comprehensive troubleshooting strategies, this protocol enables the reliable and reproducible generation of NTOs which serve as an adequate model to study relevant scientific questions. SUMMARY Three-dimensional neural tube organoids (NTOs) derived from mouse embryonic stem cells are valuable tools to study the central nervous system during early development. Here, we present a step-by-step demonstration of an optimized protocol for cultivating NTOs in vitro , providing stable culture conditions and troubleshooting strategies for reliable and reproducible NTO generation.

Tracking the spread of emerging pathogens is critical to the design of timely and effective public health responses. Policymakers face the challenge of allocating finite resources for testing and surveillance across locations, with the goal of maximising the information obtained about the underlying trends in prevalence and incidence. We model this decision-making process as an iterative node classification problem on an undirected and unweighted graph, in which nodes represent locations and edges represent movement of infectious agents among them. To begin, a single node is randomly selected for testing and determined to be either infected or uninfected. Test feedback is then used to update estimates of the probability of unobserved nodes being infected and to inform the selection of nodes for testing at the next iterations, until a certain resource budget is exhausted. Using this framework we evaluate and compare the performance of previously developed Active Learning policies, including node-entropy and Bayesian Active Learning by Disagreement. We explore the performance of these policies under different outbreak scenarios using simulated outbreaks on both synthetic and empirical networks. Further, we propose a novel policy that considers the distance-weighted average entropy of infection predictions among the neighbours of each candidate node. Our proposed policy outperforms existing ones in most outbreak scenarios, leading to a reduction in the number of tests required to achieve a certain predictive accuracy. Our findings could inform the design of cost-effective surveillance strategies for emerging and endemic pathogens, and reduce the uncertainties associated with early risk assessments in resource-constrained situations.

ABSTRACT The exogenous siRNA (exo-siRNA) pathway is a critical RNA interference response involved in controlling arbovirus replication in mosquito cells. It is initiated by the detection of viral long double-stranded RNA (dsRNA) by the RNase III enzyme Dicer 2 (Dcr2), which is processed into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs, or vsiRNAs that are taken up by the Argonaute 2 (Ago2) protein to target viral single-stranded RNAs. The detailed understanding of Dicer structure, function and domains owes much to studies outside the context of viral infection, and how Dcr2 domains contribute to detecting viral dsRNA to mount antiviral responses in infected mosquito cells remains much less understood. Here, we used a Dcr2 reconstitution system in Aedes aegypti derived Dcr2 KO cells to assess the contribution of the PAZ domain to induction of the exo-siRNA pathway following infection with Semliki Forest virus (SFV; Togaviridae , Alphavirus ). Amino acids critical for PAZ activity were identified, and loss of PAZ function affected the production of 21 nt vsiRNAs -though not the overall ability of Dcr2 to process viral dsRNA- and silencing activity. This study establishes the importance of correct vsiRNA size in mosquito exo-siRNA antiviral responses, as well as the PAZ domain’s functional contribution to Dcr2 processing of viral dsRNA to 21 nt vsiRNAs.

East, South, and Southeast Asia (together referred to as Southeastern Asia hereafter) have been recognized as critical areas fuelling the global circulation of seasonal influenza. However, the internal migration network of seasonal influenza within Southeastern Asia remains unclear, including how pandemic-related disruptions altered the network structure and circulation dynamics in this region. Here, we leveraged genetic, epidemiological, and airline travel data between 2007-2023 to characterise the multiyear dispersal patterns of influenza A/H3N2 and B/Victoria viruses both out of and within Southeastern Asia, including during seasons marked by perturbations such as the 2009 A/H1N1 and COVID-19 pandemics. We show consistent Autumn-Winter movement waves of A/H3N2 and B/Victoria from Southeastern Asia to temperate regions during interpandemic seasons. During the COVID-19 pandemic this trend was interrupted for both subtypes, however the A/H1N1 pandemic only disrupted A/H3N2 spread. For influenza strains circulating in Southeastern Asia, we find a higher persistence of A/H3N2 than B/Victoria. We find pandemic-related disruptions in A/H3N2 antigenic evolution, with a greater time-advanced antigenic evolution during the 2009 A/H1N1 pandemic, and a greater time-lagged pattern during the COVID-19 pandemic, compared to inter-pandemic levels. Internally, in comparison to the interpandemic seasons, the inferred dispersal rates within Southeastern Asia decreased by 54.7% and 79.2% during the 2009 A/H1N1 and COVID-19 pandemic seasons, respectively; further, the internal movement structure within Southeastern Asia markedly diverged during the COVID-19 pandemic season, and to a lesser extent, during the 2009 A/H1N1 pandemic season. Analyses of the trunk location and phylogenetic similarity further reveal a temporally varying pattern within Southeastern Asia, suggesting a complex source-sink network, with a notable decrease in the mixing of lineages around the COVID-19 pandemic season. Our findings provide insights into the heterogeneous interplay between influenza circulation in Southeastern Asia and two distinct pandemic-related disruptions (strong decline in human movements during the COVID-19 pandemic, pronounced pathogen interference during the A/H1N1 pandemic), which can help anticipate the effects of potential mitigation strategies and the emergence of future influenza pandemic strains on influenza dynamics.

Abstract Background : Zika virus is a small enveloped flavivirus transmitted through the bite of the aedes mosquitos. Epidemiologic survey has shown that diseases caused by Zika virus mainly include microcephaly and malformation of cranial bones.In 2016, nucleic acid screening for the zika virus was conducted in Puerto Rico, and the results indicated that the seasonal incidence rate of zika virus could reach 21.1%. Research Purpose : To investigate the effect of zika virus on the osteogenic capacity of mesenchymal stem cells. Method : We induced the zika virus-infected human mesenchymal stem cells into osteoblasts. Real-time quantitative polymerase chain reaction and Western Blot were used to detect the mRNA and protein expression of the osteogenic markers, as well as notch signaling pathway molecules. A γ-secretase inhibitor was used to explore whether the osteogenic ability of Zika virus-infected mesenchymal stem cells is restored after inhibiting the notch signaling pathway. Establish a mouse model to verify the results in vivo. Result : We found that zika virus activated the Notch signaling pathway in mesenchymal stem cells,hindering the formation of osteoblasts. After treating mesenchymal stem cells with the gamma-secretase inhibitor,the osteogenic ability of mesenchymal stem cells can be restored. The same results were observed in the mouse model. Conclusion : Zika virus suppresses the osteogenic capacity of mesenchymal stem cells via notch signaling pathway.

ABSTRACT Arthropod-borne viruses or arboviruses, including West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV) pose significant threats to public health. It is imperative to develop novel methods to control these mosquito-borne viral infections. We previously showed that insulin/insulin-like growth factor-1 signaling (IIS)-dependent activation of ERK and JAK-STAT signaling has significant antiviral activity. Continuous immune pressure can lead to adaptive mutations of viruses during infection. We aim to elucidate how IIS-signaling in mosquitoes selects for West Nile virus escape variants, to help formulate future transmission blocking strategies. We hypothesize that passage of WNV under activation of IIS will induce adaptive mutations or escape variants in the infecting virus. To test our hypothesis, WNV was serially passaged through Culex quinquefasciatus Hsu cells in the presence or absence of bovine insulin to activate IIS antiviral pressure. We sequenced WNV genes encoding for E, NS2B, NS3, and NS5 and identified variants in E and NS5 arising from IIS antiviral pressure. In parallel to the genetic analyses, we also report differences in the levels of virus replication and Akt activation in human cells using virus passaged in the presence or absence of insulin. Finally, using adult Culex quinquefasciatus , we demonstrated the enhancement of immune response gene expression in virus-infected mosquitoes fed on insulin, compared to control. Notably, virus collected from insulin-fed mosquitoes contained a non-synonymous mutation in NS3 . These results contribute towards achieving our long-term goal of manipulating mosquito IIS-dependent antiviral immunity to reduce WNV or other flavivirus transmission to mammalian hosts.

Heterochromatin is a key feature of eukaryotic genomes that serves important regulatory and structural roles in regions such as centromeres. In fission yeast, maintenance of existing heterochromatic domains relies on positive feedback loops involving histone methylation and non-coding RNAs. However, requirements for de novo establishment of heterochromatin are less well understood. Here, through a cross-based assay we have identified a novel factor influencing the efficiency of heterochromatin establishment. We determine that the previously uncharacterised protein is an ortholog of human Caprin1, an RNA-binding protein linked to stress granule formation. We confirm that the fission yeast ortholog, here named Cpn1, also associates with stress granules, and we uncover evidence of interplay between heterochromatin integrity and ribonucleoprotein (RNP) granule formation, with heterochromatin mutants showing reduced granule formation in the presence of stress, but increased granule formation in the absence of stress. We link this to regulation of non-coding heterochromatic transcripts, since in heterochromatin-deficient cells, absence of Cpn1 leads to hyperaccumulation of centromeric RNAs at centromeres. Together, our findings unveil a novel link between RNP homeostasis and heterochromatin assembly, and implicate Cpn1 and associated factors in facilitating efficient heterochromatin establishment by enabling removal of excess transcripts that would otherwise impair assembly processes.

Abstract During the period from May to November 2015, Zika virus (ZIKV) infections gained global attention in Brazil. ZIKV, a Flavivirus transmitted by Aedes mosquitoes, initially identified in 1947, has evolved from a historically inconspicuous pathogen to a significant public health concern. Beyond mild symptoms resembling dengue fever, severe conditions such as microcephaly and Guillain-Barré syndrome have been linked to ZIKV. This study focuses on epitope mapping in ZIKV non-structural proteins (NS1, NS3, and NS5), exploring the interaction with maternal and newborn immune responses through SPOT-synthesis. The objectives include establishing epitope profiles in Zika-positive individuals and correlating maternal-newborn responses. Serum samples were collected from mothers and newborns, was born in Zika Cohort Jundiai research, and Sergipe Cohort. Epitope mapping experiments was conducted using a cellulose membrane-based SPOT-synthesis technique. Notably, regions in NS1, NS3, and NS5 proteins exhibited high membrane reactivity and epitope recognition. NS1, a key antigenic marker, holds potential as an early biomarker for ZIKV detection. Additionally, NS3 and NS5 proteins displayed immunogenic potential with differential responses in newborns. The study enhances understanding of ZIKV immunology and suggests further investigations for epitope validation and potential vaccine development.

Introduction Global outbreaks of mosquito-transmitted arbovirus infections, such as dengue (DENV) and chikungunya (CHIKV), are increasing. Differentiating these infections is challenging due to non-specific symptoms and serology limitations. PCR-based approaches offer higher sensitivity and specificity. This study evaluated the performance of TaqMan™ Arbovirus Triplex Kit (ZIKV/DENV/CHIKV) (TaqMan™ Kit) to detect DENV and CHIKV in clinical samples from patients in south India. Methods In total, 280 serum samples with 90 DENV-positive, 90 CHIKV-positive, and 100 negative samples were tested with TaqMan™ Kit and CDC Trioplex Real-Time RT-PCR assay. No Zika virus was detected. The sensitivity and specificity of viral RNA detection were determined, and discordant results were resolved using comparator PCRs, dengue NS1 antigen detection, virus-specific antibody results, or previously de-identified in-house PCR results. Results The TaqMan™ Kit showed 100% agreement with the comparator for DENV detection in 92 positive samples. Among 188 samples negative for DENV by the comparator, 30 showed positive results with the TaqMan™ kit, and 23 of those were confirmed as true positives. The resulting sensitivity and specificity for DENV detection were 100% and 95.1%, respectively. For CHIKV, 77 positive and 195 negative results were concordant. Eight samples showed discordant results, but upon resolution testing, sensitivity and specificity for CHIKV were 93.9% and 100.0%, respectively. Conclusion The TaqMan™ Arbovirus Triplex Kit demonstrated high sensitivity and specificity (>93%) for detecting circulating DENV and CHIKV strains. Multiplex PCR testing can improve case detection, surveillance, and public health responses while optimizing laboratory resources for outbreak control.

Abstract Background: The Zika virus garnered global attention in 2015 due to a Brazilian epidemic, prompting a public health emergency over a surge in microcephaly cases in newborns. Long-term monitoring and care are crucial, and studies on affected children without microcephaly are essential for understanding their development. This study aims to evaluate the motor development of children over two years old exposed to the Zika virus in Jundiaí, Brazil. Method: This is a cross-sectional, comparative study integrated into the Jundiaí Zika cohort (JZC) project. The assessment instrument was the Movement Assessment Battery for Children - Second Edition (MABC-2), which measures the motor development of children and adolescents. Children with severe comorbidities and/or disorders that prevented them from doing the tasks were excluded. Results: 69 children aged 44 to 80 months completed the assessment, divided into two groups: 25 children in the Exposed group and 44 children in the Non-Exposed group. The Exposed group presented an average Total Standard Score (TSS) of 8.52 (2.97), and the Non-Exposed group presented an average TSS of 9.56 (2.97). Both groups remained in the Green Zone of the assessment, showing a satisfactory development mechanism. Conclusion: This study showed that preschool children congenitally exposed to the Zika virus without microcephaly showed satisfactory motor development compared to children in the Non-Exposed group. Trial registration: This study was approved by the Research Ethics Committee of the Faculdade de Medicina de Jundiaí (CAAE 55278022.7.0000.5412).

Abstract The increasing prevalence of tick-borne arboviral infections worldwide necessitates advanced control strategies, particularly those targeting vectors, to mitigate the disease burden. However, the cellular interactions between arboviruses and ticks, especially for negative-strand RNA viruses, remain largely unexplored. Here, we employed a proteomics informed by transcriptomics approach to elucidate the cellular response of the Rhipicephalus microplus-derived BME/CTVM6 cell line to severe fever with thrombocytopenia syndrome virus (SFTSV) infection. We generated the first de novo transcriptomes and proteomes of SFTSV- and mock-infected tick cells, identifying key host responses and regulatory pathways. Additionally, interactome analysis of the viral nucleoprotein (N) integrated host responses with viral replication mechanisms. Finally, our dsRNA-mediated gene silencing screen revealed two novel anti-SFTSV effectors, the RNA helicases, DHX9 and UPF1. Collectively, our results provide new insights into the antiviral responses of Rhipicephalus microplus vector cells and highlight critical SFTSV restriction factors, while enriching transcriptomic and proteomic resources for future research.

Primary infection with one of four dengue virus serotypes (DENV1-4) may generate antibodies that protect or enhance subsequent secondary heterotypic infections. However, the characteristics of heterotypic cross-reactive antibodies associated with protection from symptomatic infection and severe disease are not well-defined. We selected plasma samples collected before a secondary DENV heterotypic infection that was classified either as dengue fever (DF, n = 31) or dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS, n = 33) from our longstanding pediatric cohort in Nicaragua. We screened various antibody properties to determine the features correlated with protection from DHF/DSS. Protection was associated with high levels of binding of various antibody isotypes, IgG subclasses and effector functions, including antibody-dependent complement deposition, ADCD. Although the samples were derived from DENV-exposed, Zika virus (ZIKV)-naïve individuals, the protective ADCD association was stronger when assays were conducted with recombinant ZIKV antigens. Further, we showed that a complement-mediated virion lysis (virolysis) assay conducted with ZIKV virions was strongly associated with protection, a finding reproduced in an independent sample set collected prior to secondary heterotypic inapparent versus symptomatic DENV infection. Virolysis was the main antibody feature correlated with protection from DHF/DSS and severe symptoms, such as thrombocytopenia, hemorrhagic manifestations, and plasma leakage. Hence, anti-DENV antibodies that cross-react with ZIKV, target virion-associated epitopes, and mediate complement-dependent virolysis are correlated with protection from secondary symptomatic DENV infection and DHF/DSS. These findings may support the rational design and evaluation of dengue vaccines and development of therapeutics. One Sentence Summary Complement-dependent virolysis mediated by a subset of ZIKV-cross-reactive antibodies protects from symptomatic DENV infection and severe disease.

Mosquitoes are important vectors for the transmission of some major infectious diseases of humans, i.e., malaria, dengue, West Nile Virus and Zika virus. The burden of these diseases is different for different regions, being highest in tropical and subtropical areas, which have high annual rainfall, warm temperatures, and less pronounced seasonality. The life cycle of mosquitoes consists of four distinct stages: eggs, larvae, pupae, and adults. These life stages have different mortality rates and only adults can reproduce. Seasonal weather may affect the population dynamics of mosquitoes, and the relative abundance of different mosquito stages. We developed a stage-structured model that considers laboratory experiments describing how temperature and rainfall affects the reproduction, maturation and survival of different Anopheles mosquito stages, the species that transmits the parasite that causes malaria. We consider seasonal temperature and rainfall patterns and describe the stage-structured population dynamics of the Anopheles mosquito in Ain Mahbel, Algeria, Cape Town, South Africa, Nairobi, Kenya and Kumasi, Ghana. We find that neglecting seasonality leads to significant overestimation or underestimation of mosquito abundance. We find that depending on the region, mosquito abundance: peaks one, two or four times a year, periods of low abundance are predicted to occur for durations ranging from six months (Ain Mahbel) to not at all (Nairobi); and seasonal patterns of relative abundance of stages are sub-stantially different. The region with warmer temperatures and higher rainfall across the year, Kumasi, Ghana, is predicted to have higher mosquito abundance, which is broadly consistent with reported malaria deaths relative to the other countries considered by our study. Our analysis reveals distinct patterns in mosquito abundance across different months and regions. Control strategies often target one specific life stage, for example, applying larvicides to kill mosquito larvae, or spraying insecticides to kill adult mosquitoes. Our findings suggest that differences in seasonal weather affect mosquito stage structure, and that the best approaches to vector control may differ between regions in timing, duration, and efficacy.