Background While dengue NS1 antigen has been shown to be associated with disease pathogenesis in some studies, it has not been linked in other studies, with the reasons remaining unclear. NS1 antigen levels in acute dengue are often associated with increased disease severity, but there have been a wide variation in results based on past dengue infection and infecting dengue virus (DENV) serotype. As NS1 engages with many host lipids, we hypothesize that the type of NS1-lipid interactions alters its pathogenicity. Methods Primary human monocyte derived macrophages (MDMs) were co-cultured with NS1 alone or with HDL, LDL, LPS and/or platelet activating factor (PAF) from individuals with a history of past dengue fever (DF=8) or dengue haemorrhagic fever (DHF=8). IL-1β levels were measured in culture supernatants, and gene expression analysis carried out in MDMs. Monocyte subpopulations were assessed by flow cytometry. Hierarchical cluster analysis with Euclidean distance calculations were used to differentiate clusters. Differentially expressed variables were extracted and a classifier model was developed to differentiate between past DF and DHF. Results Significantly higher levels of IL-1β were seen in culture supernatants when NS1 was co-cultured with LDL (p=0.01), but with lower levels with HDL (p=0.05). MDMs of those past DHF produced more IL-1β when NS1 with PAF (p=0.02). MDMs of individuals with past DHF, were significantly more likely to down-regulate RPLP2 gene expression when macrophages were co-cultured with either PAF alone, or NS1 combined with PAF, or NS1 combined with LDL. When NS1 was co-cultured with PAF, HDL or LDL two clusters were detected based on IL10 expression, but these did not differentiate those with past DF or DHF. Conclusions As RPLP2 is important in DENV replication and in regulating cellular stress responses and immune responses and IL-10 is associated with severe disease, it would be important to further explore how differential expression of RPLP2 and IL-10 could lead to disease pathogenesis based on NS1 and lipid interactions.

ABSTRACT Background Prenatally transmitted viruses can cause severe damage to the developing brain. There is unexplained variability in prenatal brain injury and postnatal neurodevelopmental outcomes, suggesting disease modifiers. Discordant outcomes among dizygotic twins could be explained by genetic susceptibly or protection. Among several well-recognized threats to the developing brain, Zika is a mosquito-borne, positive-stranded RNA virus that was originally isolated in Uganda and spread to cause epidemics in Africa, Asia, and the Americas. In the Americas, the virus caused congenital Zika syndrome and a multitude of neurodevelopmental disorders. As of now, there is no preventative treatment or cure for the adverse outcomes caused by prenatal Zika infection. The Prenatal Infection and Neurodevelopmental Genetics (PING) Consortium was initiated in 2016 to identify factors modulating prenatal brain injury and postnatal neurodevelopmental outcomes for Zika and other prenatal viral infections. Methods The Consortium has pooled information from eight multi-site studies conducted at 23 research centers in six countries to build a growing clinical and genomic data repository. This repository is being mined to search for modifiers of virally induced brain injury and developmental outcomes. Multilateral partnerships include commitments with Children’s National Hospital (USA), Instituto Nacional de Salud (Colombia), the Natural History of Zika Virus Infection in Gestation program (Brazil), and Zika Instituto Fernandes Figueira (Brazil), in addition to the Centers for Disease Control and Prevention and the National Institutes of Health. Discussion Our goal in bringing together these sets of patient data was to test the hypothesis that personal and populational genetic differences affect the severity of brain injury after a prenatal viral infection and modify neurodevelopmental outcomes. We have enrolled 4,102 mothers and 3,877 infants with 3,063 biological samples and clinical data covering over 80 phenotypic fields and 5,000 variables. There were several notable challenges in bringing together cohorts enrolled in different studies, including variability in the timepoints evaluated and the collected clinical data and biospecimens. Thus far, we have performed whole exome sequencing on 1,226 participants. Here, we present the Consortium’s formation and the overarching study design. We began our investigation with prenatal Zika infection with the goal of applying this knowledge to other prenatal infections and exposures that can affect brain development.

Zika virus (ZIKV), a re-emerging flavivirus, is associated with devasting developmental and neurological disease outcomes particularly in infants infected in utero . Towards understanding the molecular underpinnings of the unique ZIKV disease pathologies, numerous transcriptome-wide studies have been undertaken. Notably, these studies have overlooked the assimilation of RNA-seq analysis from ZIKV-infected patients with cell culture model systems. In this study we find that ZIKV-infection of human lung adenocarcinoma A549 cells, mirrored both the transcriptional and alternative splicing profiles from previously published RNA-seq data of peripheral blood mononuclear cells collected from pediatric patients during early acute, late acute, and convalescent phases of ZIKV infection. Our analyses show that ZIKV infection in cultured cells correlates with transcriptional changes in patients, while the overlap in alternative splicing profiles was not as extensive. Overall, our data indicate that cell culture model systems support dissection of select molecular changes detected in patients and establishes the groundwork for future studies elucidating the biological implications of alternative splicing during ZIKV infection.

Over the past decades, the number of arthropod-borne virus (arbovirus) outbreaks has increased worldwide. Knowledge regarding the sylvatic cycle (i.e., non-human hosts/environment) of arboviruses is limited, particularly in Africa, and the main hosts for virus maintenance are unknown. Previous studies have shown the presence of antibodies against certain arboviruses (i.e., chikungunya-, dengue- and zika virus) in African non-human primates and bats. We hypothesize that small mammals, specifically rodents, may function as amplifying hosts in anthropogenic environments. The detection of RNA of most arboviruses is complicated by the virus’s short viremic period within their hosts. An alternative to determine arbovirus hosts is by detecting antibodies, which can persist several months. We developed a high-throughput multiplex immunoassay to detect antibodies against 15 medically relevant arboviruses. We used this assay to assess almost 1,300 blood samples of the multimammate mouse, Mastomys natalensis from Tanzania. In 24% of the samples, we detected antibodies against at least one of the tested arboviruses, with high seroprevalences of antibodies reacting against dengue virus serotype one (7.6%) and two (8.4%) and chikungunya virus (6%). Seroprevalence was higher in females and increased with age, which could be explained by inherent immunity and behavioral differences between sexes and the increased chance of exposure to an arbovirus with age. We evaluated whether antibodies against multiple arboviruses co-occur more often than randomly and found that this may be true for some members of the Flaviviridae and Togaviridae . In conclusion, the development of an assay against a wide diversity of medically relevant arboviruses enabled the analysis of a large sample collection of one of the most abundant African small mammals. Our findings suggest a role in the transmission of multiple arboviruses by this ubiquitous rodent and provide a solid foundation for future molecular screening to elucidate the role in the arbovirus transmission cycle. Author summary One of the main causes of zoonotic related human morbidity and mortality is the transmission of arthropod-borne viruses such as dengue virus, Yellow Fever virus, and chikungunya virus. These viruses cannot only infect humans but also livestock, pets, and wildlife, though our understanding of their non-human hosts remains limited. Rodents are thought to be an interesting host for these viruses because they can be abundant, often live near humans and some are already known to be viral hosts. However, research has focused on non-human primates, neglecting other potential hosts. To address this gap, we have developed a high-throughput antibody test to screen rodent blood against 15 different arboviruses. Our findings reveal that a proportion of Mastomys natalensis , a common African rodent species, carry antibodies that (cross-)react against these viruses. We hypothesize that immunologically naïve juveniles may drive transmission, particularly during population outbreaks. These outbreaks coincide with environmental conditions that are favorable for mosquitoes, the vectors of these viruses. Thus, increasing the risk of spillover to humans, livestock, and wildlife. Understanding the role of rodents in arbovirus transmission dynamics is crucial for mitigating zoonotic disease risks.

The zinc finger antiviral protein 1 (ZAP) has broad antiviral activity. ZAP is an interferon (IFN)-stimulated gene, which itself may enhance type I IFN antiviral response. In a previous study, Zika virus was identified as ZAP-resistant and not sensitive to ZAP antiviral activity. Here, we found that ZAP was associated with the inhibition of Zika virus in Vero cells, in the absence of a robust type I IFN system because Vero cells are deficient for IFN-alpha and -beta. Also, quantitative RNA-seq data indicated that endogenous ZAP is associated with altered global gene expression both in the steady state and during Zika virus infection. Further studies are warranted to elucidate this IFN-alpha and -beta independent anti-Zika virus activity and involvement of ZAP.

One of the most recent advances in the analysis of viral RNA–cellular protein interactions is the Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry (ChIRP-MS). Here, we used ChIRP-MS in mock-infected and Zika-infected wild-type cells and cells knockout for the zinc finger CCCH-type antiviral protein 1 (ZAP). We characterized “ZAP-independent” and “ZAP-dependent” cellular protein interactomes associated with flavivirus RNA and found that ZAP affects cellular proteins associated with Zika virus RNA. The ZAP-dependent interactome identified with ChIRP-MS provides potential ZAP co-factors for antiviral activity against Zika virus and possibly other viruses. Identifying the full spectrum of ZAP co-factors and mechanisms of how they act will be critical to understanding the ZAP antiviral system and may contribute to the development of antivirals.

Emerging infectious diseases are increasingly understood as a hallmark of the Anthropocene 1–3 . Most experts agree that anthropogenic ecosystem change and high-risk contact among people, livestock, and wildlife have contributed to the recent emergence of new zoonotic, vector-borne, and environmentally-transmitted pathogens 1,4–6 . However, the extent to which these factors also structure landscapes of human infection and outbreak risk is not well understood, beyond certain well-studied disease systems 7–9 . Here, we consolidate 58,319 unique records of outbreak events for 32 emerging infectious diseases worldwide, and systematically test the influence of 16 hypothesized social and environmental drivers on the geography of outbreak risk, while adjusting for multiple detection, reporting, and research biases. Across diseases, outbreak risks are widely associated with mosaic landscapes where people live alongside forests and fragmented ecosystems, and are commonly exacerbated by long-term decreases in precipitation. The combined effects of these drivers are particularly strong for vector-borne diseases (e.g., Lyme disease and dengue fever), underscoring that policy strategies to manage these emerging risks will need to address land use and climate change 10–12 . In contrast, we find little evidence that spillovers of directly-transmitted zoonotic diseases (e.g., Ebola virus disease and mpox) are consistently associated with these factors, or with other anthropogenic drivers such as deforestation and agricultural intensification 13 . Most importantly, we find that observed spatial outbreak intensity is primarily an artefact of the geography of healthcare access, indicating that existing disease surveillance systems remain insufficient for comprehensive monitoring and response: across diseases, outbreak reporting declined by a median of 32% (range 1.2%-96.7%) for each additional hour’s travel time from the nearest health facility. Our findings underscore that disease emergence is a multicausal feature of social-ecological systems, and that no one-size-fits-all global strategy can prevent epidemics and pandemics. Instead, ecosystem-based interventions should follow regional priorities and system-specific evidence, and be paired with investment in One Health surveillance and health system strengthening.

Abstract Zika virus (ZIKV) garnered global attention over the past decade as outbreaks of the disease were linked to neurological complications. There are currently no antiviral drugs or vaccines with proven efficacy. The identification of human proteins targeted by ZIKV is crucial for deciphering the host pathways hijacked by the virus to replicate, evade innate immunity, and induce neuropathogenesis. In our study, Y2H screening of 8 ZIKV proteins identified 85 interacting host factors. These host factors are primarily involved in immune and inflammatory responses, cell death, centrosome and cell cycle regulation, ubiquitin pathways, central nervous system (CNS) development and neurological disorders. Rho associated coiled-coil containing protein kinase 2 (ROCK2) was selected for further evaluation of its role in ZIKV infection. Immunofluorescence staining showed colocalization between ROCK2 and ZIKV NS5 in the cell nucleus, indicating the interaction likely contributes to viral replication via immune and inflammatory responses. Further siRNA knockdown of ROCK2 resulted in significant inhibition of ZIKV genome copy number. The ZIKV-host interactome was also used as a platform to identify druggable human proteins targeted by existing host-directed antiviral drugs. Taken together, findings of this study aid in improving the understanding of ZIKV pathogenesis and identifying potential therapeutic targets.

Background: Paediatric neuroblastoma and brain tumours account for a third of all childhood cancer-related mortality. High-risk neuroblastoma is highly aggressive and survival is poor despite intensive multi-modal therapies with significant toxicity. Novel therapies are desperately needed. The Zika virus (ZIKV) can access the nervous system and there is growing interest in employing ZIKV as a potential therapy against paediatric nervous system tumours, including neuroblastoma. Methods Here, we perform extensive data mining, integration and re-analysis of ZIKV infection datasets to highlight molecular mechanisms that may govern the oncolytic response in neuroblastoma cells. We collate infection data of multiple neuroblastoma cell lines by different ZIKV strains from a body of published literature to inform the susceptibility of neuroblastoma to the ZIKV oncolytic response. Integrating published transcriptomics, interaction proteomics, dependency factor and compound datasets we propose the involvement of multiple host systems during ZIKV infection. Results Through data mining of published literature, we observed most paediatric neuroblastoma cell lines to be highly susceptible to ZIKV infection and propose the PRVABC59 ZIKV strain to be the most promising candidate for neuroblastoma oncolytic virotherapy. ZIKV induces TNF signalling, lipid metabolism, the Unfolded Protein Response (UPR), and downregulates cell cycle and DNA replication processes. ZIKV infection is dependent on sterol regulatory element binding protein (SREBP)-regulated lipid metabolism and three protein complexes; V-ATPase, ER Membrane Protein Complex (EMC) and mammalian translocon. We propose ZIKV non-structural protein 4B (NS4B) as a likely mediator of ZIKVs interaction with IRE1-mediated UPR, lipid metabolism and mammalian translocon. Conclusions Our work provides a significant understanding of ZIKV infection in neuroblastoma cells, which will facilitate the progression of ZIKV-based oncolytic virotherapy through pre-clinical research and clinical trials.

ABSTRACT Background Gestational Zika virus (ZIKV) infection is associated with the development of congenital Zika syndrome (CZS), which includes microcephaly and fetal demise. The magnitude and quality of orthoflavivirus-specific humoral immunity have been previously linked to the development of CZS. However, the role of ZIKV NS1-specific humoral immunity in mothers and children with prenatal ZIKV exposure and CZS remains undefined. In addition, considering that most of the at-risk population lives in dengue virus (DENV)-endemic areas, it is not clear what is the association between pre-existing DENV NS1-specific humoral immunity and CZS. Methods Here, we studied 328 mothers and children with a clinical diagnosis and seropositivity for ZIKV infection during pregnancy, included during the 2015-2016 ZIKV epidemic in Colombia. We also performed clinical evaluation and pediatric neurological follow-up. The relative levels of circulating NS1-specific IgM and IgG against ZIKV and DENV were evaluated in mothers and children, and the association with the development of microcephaly was analyzed. Results DENV and ZIKV IgG-NS1 antibodies in pregnant women were placentally transferred, and this passage and its duration in children depended on the maternal levels of the antibodies. We reported that higher concentrations of pre-existing DENV, but not ZIKV IgG-NS1 antibodies, were associated with a reduced risk of CZS-related microcephaly. Also, we observed that the IgM-NS1 response presents as long-term and has a minor association with poor outcomes. Conclusions The development of microcephaly in children prenatally exposed to ZIKV is associated with low plasma levels of placentally transferred, pre-existing DENV IgG-NS1 antibodies. These data are compatible with a protective role of anti-NS1 IgG antibodies against ZIKV infection during pregnancy and highlight the promising role of NS1 as an orthoflavivirus vaccine target in high-risk populations.

Abstract After the Zika virus (ZIKV) epidemic in Brazil, ZIKV infections were linked to damage to the central nervous system (CNS) and congenital anomalies. Due to the virus’s ability to cross the placenta and reach brain tissue, its effects become severe, leading to Congenital Zika Syndrome (CZS) and resulting in neuroinflammation, microglial activation, and secretion of neurotoxic factors. The presence of ZIKV triggers an inadequate fetal immune response, as the fetus only has the protection of maternal antibodies of the Immunoglobulin G (IgG) class, which are the only antibodies capable of crossing the placenta. Because of limited understanding regarding the long term consequences of ZIKV infection and the involvement of maternal antibodies, this study sought to assess the impact of the ZIKV+IgG⁺complex on murine microglial cells. The cells were exposed to ZIKV, IgG antibodies, and the ZIKV+IgG⁺complex for 24 and 72 hours. Treatment-induced cytotoxic effects were evaluated using the cell viability assay, oxidative stress, and mitochondrial membrane potential. The findings indicated that IgG antibodies exhibit cytotoxic effects on microglia, whether alone or in the presence of ZIKV, leading to compromised cell viability, disrupted mitochondrial membrane potential, and heightened oxidative damage. Our conclusion is that IgG antibodies exert detrimental effects on microglia, triggering their activation and potentially disrupting the creation of a neurotoxic environment. Moreover, the presence of antibodies may correlate with an elevated risk of ZIKV-induced neuroinflammation, contributing to long-term CNS damage.

Abstract Background Arbovirus infection outbreaks are becoming more common in Africa. However, it is still difficult and crucial to better understand arbovirus transmission patterns, disease trends, and burdens. The epidemiology of these infections—dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), West Nile virus (WNV), Rift Valley fever virus (RVFV), and yellow fever virus (YFV)—is unfortunately not well understood. This review provides an epidemiological inventory of DENV, ZIKV, CHIKV, WNV, RVFV, and YFV infections in Africa, with helpful results for risk mapping and upcoming prevention and control initiatives. Methods This systematic review protocol implements the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and an expert-evaluated design and laboratory assay and reporting evaluation (DARE) concept. Two independent reviewers conducted preliminary literature searches in PubMed in May 2023 to improve the search keywords, strategy, and inclusion criteria while considering the context and scientific significance. The final search will be conducted using PubMed, ScienceDirect (SCOPUS), the Web of Science Core Collection, African Journal Online and Google Scholar. Two reviewers will simultaneously and independently conduct searches, screen studies, and extract data. Quality assessment will be performed by two independent epidemiology experts, and discrepancies will be handled by consensus or by consulting a third reviewer. Meta-analysis will be performed to determine the pooled estimates of arbovirus circulation and transmission patterns in Africa. Discussion In this review, we present an epidemiological inventory with information that will be relevant for risk assessment, future arbovirus infection outbreak prevention, and arbovirus infection outbreak control in Africa. This will include estimating the patterns, trends, and burdens of arboviral infection across Africa, as well as identifying the regions with the highest risk of transmission. This approach will be crucial for developing well-informed policies for epidemic prevention. Systematic review registration The review is registered and accessible at Prospero with the registration ID CRD42023434939.

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

Abstract Background The high frequency of Zika virus (ZIKV) infection prompted the World Health Organization (WHO) to declare it an emerging threat in 2016. Presently, countries in South America continue to report a significant number of Zika virus cases. Zika virus infection has been associated with neurological diseases. This article explores the impact of ZIKV infection on IgG + Zika-reactive conditions using a neurosphere model. Methods Neurospheres derived from Wistar rats were exposed to Zika virus and IgG + Zika-reactive conditions. The study assessed the area, irregularity rate, and relative gene expression of NOTCH-1, HES-1, and HEY-1, as well as the expression of GCSF and IL-10. Results The irregularity rate of ZIKV neurospheres decreased at its lowest concentration; however, our morphology analysis was insufficient to fully elucidate the impact of ZIKV on neurospheres. Interestingly, IgG + serum exhibited neuroprotective effects against subsequent Zika virus exposure, restoring NOTCH-1 and HES-1 expression levels to normal. HEY-1 expression remained unaffected by Zika virus exposure but decreased with IgG + serum. Surprisingly, levels of the anti-inflammatory markers GCSF and IL-10 showed no significant changes. Conclusions These findings highlight the complex interplay between ZIKV infection, immune response, and neurodevelopmental processes. Further research is necessary to elucidate the precise mechanisms underlying these observations and explore potential therapeutic interventions.

The Advisory Committee on Immunization Practices (ACIP) recommended that dengue pre-vaccination screening tests for Dengvaxia administration have at least 98% specificity and 75% sensitivity. This study evaluates the performance of commercial anti-DENV IgG tests to identify tests that could be used for pre-vaccination screening. First, for 7 tests, we evaluated sensitivity and specificity in early convalescent dengue virus (DENV) infection, using 44 samples collected 7-30 days after symptom onset and confirmed by RT-PCR. Next, for the 5 best performing tests and two additional tests (with and without an external test reader) that became available later, we evaluated performance to detect past dengue infection among a panel of 44 specimens collected in 2018-2019 from healthy 9-16-year-old children from Puerto Rico. Finally, a full-scale evaluation was done with the 4 best performing tests using 400 specimens from the same population. We used virus focus reduction neutralization test and an in-house DENV IgG ELISA as reference standards. Of seven tests, five showed ≥75% sensitivity detecting anti-DENV IgG in early convalescent specimens with low cross-reactivity to Zika virus. For the detection of previous DENV infections the tests with the highest performance were the Euroimmun NS1 IgG ELISA (sensitivity 84.5%, specificity 97.1%) and CTK Dengue IgG rapid test R0065C with the test reader (sensitivity 76.2% specificity 98.1%). There are IgG tests available that can be used to accurately classify individuals with previous DENV infection as eligible for dengue vaccination to support safe vaccine implementation.

ABSTRACT Mosquito-borne Zika virus (ZIKV; orthoflavivirus, Flaviviridae ) has become a global health problem due to expansion of the geographic distribution of Asian Lineage virus. Contemporary ZIKV strains of African lineage have recently gained increased attention due to their epidemic potential and their capacity to be highly teratogenic in humans. The ZIKV non-structural NS1 protein from recent West African strains Africa was been studied where with view of its importance in the pathogenicity. NS1 protein from contemporary West African ZIKV (NS1 CWA ) and historical African ZIKV strain MR766 (NS1 MR766 ) differ by seven amino-acid substitutions. Expression of recombinant NS1 proteins showed differences in the subcellular distribution between NS1 CWA and NS1 MR766 in HEK-293T cells. There was an increased secretion efficiency of soluble NS1 CWA compared to NS1 MR766 . The replication of a chimeric MR766/NS1 CWA virus was studied in Vero and A549 cells. Insertion of NS1 CWA into MR766 enhances virus replication in both cell lines leading to more pronounced cell death. This correlated with lower up-regulation of IFN-β and interferon-stimulated gene mRNA in A549 cells infected by MR766/NS1 CWA virus. Our data raise the question on the importance of NS1 protein in the pathogenicity of contemporary ZIKV from West Africa, and point to differences within viral strains belonging to the same African lineage. AUTHOR SUMMARY Mosquito-borne Zika virus (ZIKV) of African lineage has the potential to cause epidemic along with a high risk of fetal pathogenicity. Too little is still known on the features of contemporary ZIKV from West Africa. We find there is a remarkable conservation of NS1 amino-acid residues between ZIKV strains recently isolated in Senegal and Guinea. Analysis of recombinant ZIKV NS1 protein revealed efficient secretion of contemporary African NS1 protein from human cells. Using infectious molecular clone of African ZIKV, we showed that contemporary West Africa NS1 protein influences virus replication and innate immune activation. The NS1 protein has been proposed as playing a major role in the pathogenicity of contemporary ZIKV from West Africa.

Abstract Previous studies about vector-borne diseases have emphasized the feedback between human psychology and diseases but neglected the changes in psychological processes. Here I first studied whether and how the two types of psychological dynamics in people’s Terror-To-Death (TTD) — periodical terror reinforcement and memory decay of terror — can influence the host-vector-pathogen interactions. Through developing a generic Ross-MacDonald model with TTD dynamics tailored for Zika virus transmitted by Aedes aegypti mosquito, I found that in general, the increase in initial terror increases control effort, while memory decay of terror decreases disease control. Memory decay also exhibits a threshold effect: when initial terror is below certain level, TTD decay would not influence the system much; once initial terror reaches a threshold, memory decay of TTD can largely reduce the public’s control effort, increase mosquito population and disease level in the system under a larger mosquitoes’ carrying capacity. Adding periodical terror reinforcement could introduce dynamical oscillation to the system, dampen the peak of human infection, and shorten the time of disease outbreak. If the reinforcement frequency is large enough, system dynamics could approach the scenario with constant TTD in the absence of memory decay. This work significantly advances the theory in disease epidemiology and biopsychology and can provide guidance for disease control by considering the joint effects of initial terror, the public’s memory decay, and the frequency of terror reinforcement simultaneously.

Mosquito-borne viruses cause more than 400 million annual infections and place over half of the world’s population at risk. Despite this importance, the mechanisms by which arboviruses infect the mosquito host and disseminate to tissues required for transmission are not well understood. Here, we provide evidence that mosquito immune cells, known as hemocytes, play an integral role in the dissemination of dengue virus (DENV) and Zika virus (ZIKV) in the mosquito Aedes aegypti . We establish that phagocytic hemocytes are a focal point for virus infection and demonstrate that these immune cell populations facilitate virus dissemination to the ovaries and salivary glands. Additional transfer experiments confirm that virus-infected hemocytes confer a virus infection to non-infected mosquitoes more efficiently than free virus in acellular hemolymph, revealing that hemocytes are an important tropism to enhance virus dissemination in the mosquito host. These data support a “trojan horse” model of virus dissemination where infected hemocytes transport virus through the hemolymph to deliver virus to mosquito tissues required for transmission and parallels vertebrate systems where immune cell populations promote virus dissemination to secondary sites of infection. In summary, this study significantly advances our understanding of virus infection dynamics in mosquitoes and highlights conserved roles of immune cells in virus dissemination across vertebrate and invertebrate systems.

Abstract Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. To investigate whether USUV is able to infect the brain similarly to WNV and ZIKV, we determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-β and 2’C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers. USUV and ZIKV reached comparable titers and all three viruses primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-β inhibited replication of the arboviruses, while 2’C-methyl-cytidine reduced titers of USUV and ZIKV, but not WNV. Collectively, USUV can infect human brain tissue, showing similarities in replication, tropism and neurovirulence as WNV and ZIKV. Further, this model system can be applied as a preclinical model to determine the efficacy and safety of drugs to treat viral infections of the brain.

Zika virus (ZIKV) is a notable arbovirus with maternal, sexual, and TORCH-related transmission capabilities. After 2015, Brazil had the highest number of ZIVK-infected pregnant women who lost their babies or delivered them with Congenital ZIKV Syndrome (CZS). ZIKV infection has been linked to variations in endogenous retroviruses (ERVs) and interleukins within the placenta. ERVs, remnants of ancient viral infections integrated into the genome, include syncytin receptors crucial for placental development. Interleukins, immune response regulators, aid immune tolerance and support syncytial structure development in the placenta, where syncytin receptors facilitate vital cell-to-cell fusion events. Since ZIKV can infect trophoblast cells, we investigated the relationship between ZIKV infections and ERV and interleukin modulations. Investigating the impact of ZIKV on trophoblast cells, we examined two cell types (BeWo and HTR8) infected with ZIKV-MR766 (African) and ZIKV-IEC-Paraíba (Asia-Brazilian) using Taqman and RT2 Profiler PCR Array assays. While early ZIKV infection (24-48 hours) did not induce differential ERV and interleukin expression, future studies involving additional trophoblastic cell lineages and extended infection timelines will illuminate the dynamic interplay between ZIKV, HERVs, interleukins, and cytokines essential for placental formation and function.

Background Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, barriers to broader take-up include the need for high sequencing depths, long turnaround times, and limited sensitivity. Newer metagenomics protocols based on Oxford Nanopore Technologies (ONT) sequencing allow acquisition and analysis of data in real time, potentially reducing the need for high-volume sequencing and enabling point-of-care testing. Furthermore, targeted approaches that selectively amplify known pathogens could improve sensitivity. Methods We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Biosciences Comprehensive Viral Research Panel (VRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers. Results Capture with the Twist VRP increased sensitivity by at least 10-100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600-6,000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the VRP and ONT. Conclusions Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist VRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.

The 2016 outbreak of Zika virus (ZIKV) infected millions and resulted in thousands of infants born with malformations. Though the clusters of severe birth defects resulting from this outbreak have subsided, ZIKV continues to be a concern throughout much of Latin America and the Caribbean. Travel and sexual intercourse remain the dominant transmission risk factors for women of reproductive age and their partners. This is particularly true for communities in Brooklyn, New York, that comprise large immigrant and foreign-born populations. Practitioners of public health understand little about how women at risk for ZIKV are most likely to receive information about the virus or who they trust most to provide that information. In the context of five focus group discussions, this study explored the knowledge and communication preferences of 20 women of reproductive age in Central Brooklyn. Results derived from a thematic analysis suggest that while most women are familiar with mosquitos as ZIKV vectors, knowledge of sexual transmission is considerably lower. Many respondents believe that only women who are pregnant or trying to become pregnant are at risk, and public health agencies, such as the U.S. Centers for Disease Control and Prevention, remain the most trusted sources of information. These findings can support more effective communication about the risks of ZIKV infection and other vector-borne diseases to women in New York City and similar urban communities.

SUMMARY Subgenomic flavivirus RNAs (sfRNAs) are structured RNA elements encoded in the 3’-UTR of flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze the production of sfRNAs using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) and super-resolution microscopy during West Nile virus, Zika virus, or Dengue virus serotype 2 infection. We show that sfRNAs are initially localized diffusely in the cytosol or in processing bodies (P-bodies). However, upon activation of the host antiviral endoribonuclease, Ribonuclease L (RNase L), nearly all sfRNAs re-localize to antiviral biological condensates known as RNase L-induced bodies (RLBs). RLB-mediated sequestration of sfRNAs reduces sfRNA association with RNA decay machinery in P-bodies, which coincides with increased viral RNA decay. These findings establish a role of RLBs in promoting viral RNA decay, demonstrating the complex host-pathogen interactions at the level of RNA decay and biological condensation. Highlights Single-molecule imaging of sfRNA production and localization sfRNAs localize to RNase L-induced bodies RNase L-induced bodies sequester sfRNAs away from P-bodies Sequestration of sfRNAs by RNase L-induced bodies enhances decay of viral genomes

Unbiased long read sequencing holds enormous potential for the detection of pathogen sequences in clinical samples. However, the untargeted nature of these methods precludes conventional PCR approaches, and the metagenomic content of each sample increases the challenge of bioinformatic analysis. Here, we evaluate a previously described novel workflow for unbiased RNA virus sequence identification in a series of contrived and real-world samples. The novel multiplex library preparation workflow was developed for the Oxford Nanopore Technologies (ONT) MinION TM sequencer using reverse transcription, whole genome amplification, and ONT’s Ligation Sequencing Kit with Native Barcode Expansion. The workflow includes spiked MS2 Phage as an internal positive control and generates an 8-plex library with 6 samples, a negative control and a gfp transcript positive control. Targeted and untargeted data analysis was performed using the EPI2ME Labs framework and open access tools that are readily accessible to most clinical laboratories. Contrived samples composed of common respiratory pathogens (Influenza A, Respiratory Syncytial Virus and Human Coronavirus 229E) in viral transport media (VTM) and bloodborne pathogens (Zika Virus, Hepatitis A Virus, Yellow Fever Virus and Chikungunya Virus) in human plasma were used to establish the limits of detection for this assay. We also evaluated the diagnostic accuracy of the assay using remnant clinical samples and found that it showed 100% specificity and 62.9% clinical sensitivity. More studies are needed to further evaluate pathogen detection and better position thresholds for detection and non-detection in various clinical sample metagenomic mixtures.

ABSTRACT Zika virus (ZIKV) have become a global health problem over the past decade due to the extension of the geographic distribution of ZIKV of Asian genotype. Epidemics of Asian ZIKV have been associated with developmental disorders in humans. ZIKV of African lineage would have an epidemic potential associated to fetal pathogenicity requiring a greater attention towards the most recently isolated viral strains from West Africa. In the present study, an infectious molecular clone GUINEA-18 has been obtained from viral strain ZIKV-15555 that had been sequenced from an individual infected by ZIKV in Guinea in 2018. A molecular clone-based comparative study between GUINEA-18 and viral clone MR766 MC from historical African ZIKV strain MR766 revealed a lower replication rate for GUINEA-18 associated to a weaker cytotoxicity and reduced innate immune system activation in Vero E6, A549 and HCM3 cell lines. Analysis of chimeric viruses between MR766 MC and GUINEA-18 stressed the importance NS1/NS4B proteins with a particular focus for NS4B on GUINEA-18 replication properties. ZIKV has developed strategies to prevent cytoplasmic stress granule formation which occurs in response to virus infection. Study of G3BP protein showed that GUINEA-18 but not MR766 MC was efficient to inhibit stress granule assembly in A549 cells subjected to a physiological stressor. GUINEA-18 depends on NS1/NS4B proteins for suppressing stress granule response to environmental stress. The involvement of GUINEA-18 NS1/NS4B proteins on virus replication capability and host-cell responses to ZIKV infection raises the question of the importance of nonstructural proteins in the pathogenicity of contemporary viral strains from West Africa. AUTHOR SUMMARY Most of studies having for objectives to understand the biology of Zika virus (ZIKV) were carried out using epidemic viral strains of Asian lineage. It is now admitted that ZIKV of African genotype would have also a great epidemic potential associated a high risk of fetal pathogenicity. Today, it is urgent to improve our knowledge on recently isolated ZIKV strains in West Africa. In our study, we used the sequence of viral strain from an individual infected by ZIKV in Guinea in 2018 to generate an infectious molecular clone. Analysis of viral clone highlighted the preponderant role of NS1/NS4B proteins in virus replication strategy and cell interactions with a particular focus on ZIKV-specific stress granule formation blockade. We believe that our data will improve our knowledge on the biology of contemporary West Africa ZIKV opening perspectives towards a better understanding on the pathogenicity of African viral strains.