Infection by flaviviruses leads to dramatic remodeling of the endoplasmic reticulum (ER). Viral replication occurs within virus-induced vesicular invaginations in the ER membrane. A hallmark of flavivirus infection is expansion of the ER membrane which can be observed at specific time points post infection. However, this process has not been effectively visualized in living cells throughout the course of infection at the single cell resolution. In this study, we developed a plasmid-based reporter system to monitor flavivirus infection and simultaneous virus-induced manipulation of single cells throughout the course of infection in real-time. This system requires viral protease cleavage to release an ER-anchored fluorescent protein infection reporter that is fused to a nuclear localization signal (NLS). This proteolytic cleavage allows for the translocation of the infection reporter signal to the nucleus while an ER-specific fluorescent marker remains localized in the lumen. Thus, the construct allows for the visualization of virus-dependent changes to the ER throughout the course of infection. In this study, we show that our reporter was efficiently cleaved upon the expression of multiple flavivirus proteases, including dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). We also found that the DENV protease-dependent cleavage of our ER-anchored reporter exhibited more stringent cleavage sequence specificity than what has previously been shown with biochemical assays. Using this system for long term time-lapse imaging of living cells infected with DENV, we observed nuclear translocation of the reporter signal beginning approximately 8 hours post-infection, which continued to increase throughout the time course. Interestingly, we found that increased reporter signal translocation correlated with increased ER signal intensity, suggesting a positive association between DENV infection and ER expansion in a time-dependent manner. Overall, this report demonstrates that the FlavER platform provides a useful tool for monitoring flavivirus infection and simultaneously observing virus-dependent changes to the host cell ER, allowing for study of the temporal nature of virus-host interactions.

Despite the availability of vaccines and approved therapeutics, the COVID-19 pandemic continues to rise owing to the emergence of newer variants. Several multi-omics studies have made available extensive evidence on host-pathogen interactions and potential therapeutic targets. Nonetheless, an increased understanding of host signaling networks regulated by post-translational modifications and their ensuing effect on the biochemical and cellular dynamics is critical to expanding the current knowledge on the host response to SARS-CoV-2 infections. Here, employing unbiased global transcriptomics, proteomics, acetylomics, phosphoproteomics, and exometabolome analysis of a lung-derived human cell line, we show that SARS-CoV-2 Norway/Trondheim-S15 strain induces time-dependent alterations in the induction of type I IFN response, activation of DNA damage response, dysregulated Hippo signaling, among others. We provide evidence for the interplay of phosphorylation and acetylation dynamics on host proteins and its effect on the altered release of metabolites, especially organic acids and ketone bodies. Together, our findings serve as a resource of potential targets that can aid in designing novel host-directed therapeutic strategies.

The world has seen numerous infectious disease outbreaks in the past decade. In many cases these outbreaks have had considerable perinatal health consequences including increased risk of preterm delivery (e.g., influenza, measles, and COVID-19), and the delivery of low birth weight or small for gestational age babies (e.g., influenza, COVID-19). Furthermore, severe perinatal outcomes including perinatal and infant death are a known consequence of multiple infectious diseases (e.g., Ebola virus disease, Zika virus disease, pertussis, and measles). In addition to vaccination during pregnancy (where possible), pregnant women, are provided some level of protection from the adverse effects of infection through community-level application of evidence-based transmission-control methods. This review demonstrates that it takes almost two years for the perinatal impacts of an infectious disease outbreak to be reported. However, many infectious disease outbreaks between 2010 and 2020 have no associated pregnancy data reported in the scientific literature, or pregnancy data is reported in the form of case-studies only. This lack of systematic data collection and reporting has a negative impact on our understanding of these diseases and the implications they may have for pregnant women and their unborn infants. Monitoring perinatal health is an essential aspect of national and global healthcare strategies as perinatal life has a critical impact on early life mortality as well as possible effects on later life health. The unpredictable nature of emerging infections and the potential for adverse perinatal outcomes necessitate that we thoroughly assess pregnancy and perinatal health implications of disease outbreaks and their public health interventions in tandem with outbreak response efforts. Disease surveillance programs should incorporate perinatal health monitoring and health systems around the world should endeavour to continuously collect perinatal health data in order to quickly update pregnancy care protocols as needed.

The flavivirus family are responsible for the most abundant arboviral diseases of humans in terms of geographical distribution, morbidity and mortality; at least 2.5 billion people are at risk with, for example, an estimated 100-400 million Dengue infections a year. However, for infections by Dengue, Zika or Yellow Fever virus there are no effective anti-infective drug treatments nor for Dengue or Zika virus a safe effective vaccine and prevention at present focusses on vector (mosquito) control. Whilst symptoms from Dengue, Zika and Yellow Fever virus infection may be mild for some, they are very serious and life threatening for others. For instance, severe Dengue is a leading cause of hospitalisation and death among children and adults in Asian and Latin American countries. Likewise, Zika infection can have catastrophic consequences for pregnant women following the passing of the virus to their foetus with arising miscarriage or birth defects including microcephaly that can be fatal. The aim of the present study was to evaluate the potential of a unique low molecular weight dextran sulphate (LMW-DS) clinical stage drug, ILB ® , to inhibit infection of human cells by four serotypes of Dengue virus (DENV1-4), two strains of Zika virus (African and Asian) and Yellow Fever virus (vaccine strain YF17D) assessed by immunofluorescence of viral particles. ILB ® potently inhibited infection by all the strains of Dengue, Zika and Yellow Fever virus in a concentration-dependent manner with IC 50 for ILB® ranging from 31 to 343 μg/ml. In conclusion, given the safety profile of ILB ® established in a number of Phase I and Phase II clinical trials, these results highlight the potential of ILB ® to treat patients infected with Dengue, Zika or Yellow Fever virus with the opportunity to translate the findings quickly by clinical investigation.

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFNε) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs α, β and λ. We show the necessity of IFNε for Zika Virus (ZIKV) protection by: increased susceptibility of IFNε -/- mice; their “rescue” by intravaginal recombinant IFNε treatment and blockade of protective endogenous IFNε by neutralising antibody. Complementary studies in human FRT cell lines showed IFNε had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNε activated STAT1/2 pathways similar to IFNα and λ that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFNε exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFNε. However, the IFNε expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNβ or λ. Thus, the constitutive expression of IFNε provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFNε provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy. Author Summary The female reproductive tract (FRT) is vulnerable to sexually transmitted infections and therefore a well-tuned immune surveillance system is crucial for maintaining a healthy FRT. However, our understanding of the factors that impact viral infection of the FRT and the host response are not well understood. In this work we investigate the role of a hormonally regulated type I interferon, IFN epsilon (IFNε) in control of Zika virus (ZIKV) infection of the FRT. IFNε is unique compared to other canonical type-I IFNs in that it is constitutively expressed by epithelial cells of the FRT with expression levels controlled by progesterone and not in response to viral infection. We demonstrate that IFNε has anti-ZIKV properties using a combination of IFNε KO mice, blockade of endogenous IFNε by neutralising Abs and rescue of IFNε KO mice by recombinant IFNε administered directly to the FRT. Furthermore, we complemented our in vivo studies using human FRT derived cell lines. Importantly, ZIKV NS proteins did not block IFNε expression despite their ability to antagonise the expression of IFNβ or λ. Collectively this work implicates IFNε as a key type-I IFN that provides a distinct homeostatic antiviral environment in the FRT.

Background: COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. Methods: A ligation-based typing assay was developed to detect know variants of SARS-CoV-2 by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time PCR instrumentation for assay performance. Findings: The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold-standard of viral genome sequencing. It achieved an average limit of detection of 7.4 x 10 4 genome copies per mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by sequence alignment of clinical samples. Interpretation: The assay demonstrates potential for robust variant-typing with performance comparable to next generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource-dependency and generalizability could expand access to variant classification information for pandemic surveillance. Funding Information: US National Institutes of Health R01 (AI157827) (JES and FRH) and Centers for Disease Control and Prevention (75D30121C10094) (JES and SRD) funded the study. DJN thanks the National Science Foundation Graduate Research Fellowship (1937963). Declaration of Interests: There are no conflicts of interest to declare. Ethics Approval Statement: All specimens were collected, de-identified, and transferred from Vanderbilt University Medical Center as approved by the Institutional Review Board (IRB #201708 and #201804).

Abstract Mosquitoes are vectors of several arboviruses belonging to the genus Alphavirus and Flavivirus . Costa Rica is endemic for several arboviruses. To described and analyze the community structure of vectors in the country, a sampling was performed in two different coastal locations with evidence of arboviral activity during rainy and dry seasons. Encephalitis vector surveillance Traps, CDC Female Gravid Traps and ovitraps were used. Viral detection for several arbovirus was attempted. Blood-meal identification was also performed. A total of 1802 adult mosquitoes belonging to 55 species were captured. Culex quinquefasciatus was the most captured. species The Normalized Difference Vegetation Index (NDVI) was estimated for each area during rainy and dry season. The higher difference between seasonal regional NDVI yield lower values of Chao- Sørensen Similarity Index. Chao2 values and Shannon Index for species diversity were also estimated. There was no viral detection for VEEV, WNV, Madariaga. Dengue virus and Zika virus were detected in two separate Culex quinquefasciatus . The main sources of bloodmeal chickens (60%) and humans (27.5%). Both sampled areas have different seasonal dynamics and population turnover reflected in the Chao2 species richness estimation values and Shannon diversity Index. NDVI influence the mosquito diversity at a regional scale more than at a local scale. However, yearlong continuous sampling is required for a better understanding of local dynamics.

ABSTRACT Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, production of Zika virus has been successful from CMV-promoter driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued virus maintained similar replication kinetics in several cell lines commonly used in flavivirus research. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.

An in-depth examination of people’s risk perceptions under public health emergencies is critical for risk management and policymaking. Based on the first-hand data collected in the early outbreak of coronavirus pneumonia (COVID-19), the paper attempts to explore the characteristics, heterogeneity, and influencing factors of risk perceptions in China. We found that during that period, individuals exhibited a higher risk perception of COVID-19, which may be classified into perceived severity and vulnerability. Second, there was significant social and spatial heterogeneity in risk perceptions. The internal differences are most pronounced in the social groups of age, education status, self-rated health, and employment status. Respondents inside and outside of Hubei province expressed similar perceptions of vulnerability but differed in their perceptions of severity. Third, apart from aforementioned internal(individual-) factors, we found that external (contextual-) factors can significantly influence public risk perceptions, including residential environment (such as housing condition, neighborhood type, medical resources provision, etc.), satisfaction with information, and risk exposure to COVID-19. The theoretical and practical implications of this study enrich our knowledge of the epidemic’s risk perception and provide specific suggestions for preventing the local spread of this ongoing epidemic.

Neuronal progenitor subtypes have distinct fate restrictions regulated by time-dependent activation of energetic pathways. Thus, the hijacking of cellular metabolism by Zika virus (ZIKV) to support its replication may contribute to damage in the developing fetal brain. Here, we showed that ZIKV replicates differently in two glycolytically distinct hiPSC-derived neuronal progenitors that correspond to early and late progenitors in the forebrain. This differential replication alters the transcription of metabolic genes and upregulates the glycolytic capacity of progenitor subtypes. Analysis using Imagestream® revealed that, during early stages of infection, ZIKV replication in early progenitors increases lipid droplet abundance and decreases mitochondrial size and membrane potential. During later stages infection, early progenitors show increased subcellular distribution of lipid droplets, whilst late progenitors show decreased mitochondria size. The finding that there are hi-NPC subtype-specific alterations of cellular metabolism during ZIKV infection may help to explain the differences in brain damage over each trimester.

Insect gut microbiota plays important roles in host physiology, such as nutrition, digestion, development, fertility, and immunity. We have found that in the intestine of Aedes aegypti , SLIMP (seryl-tRNA synthetase like insect mitochondrial protein) knockdown followed by a blood meal promotes dysbiosis, characterized by the overgrowth of a specific bacterial phylum, Bacteroidetes. In turn, the latter decreased both infection rates and Zika virus prevalence in the mosquitoes. Previous work in Drosophila melanogaster showed that SLIMP is involved in protein synthesis and mitochondrial respiration in a network directly coupled to mtDNA levels. There are no other reports on this enzyme and its function in other insect species. Our work expands the knowledge of the role of these SerRS paralogs. We show that A. aegypti SLIMP (AaeSLIMP) clusters with SLIMPs of the Nematocera sub-order, which have lost both the tRNA binding domain and active site residues, rendering them unable to activate amino acids and aminoacylate tRNAs. Knockdown of AaeSLIMP did not significantly influence the mosquitoes’ survival, oviposition, or eclosion. It also neither affected midgut cell respiration nor mitochondrial ROS production. However, it caused dysbiosis, which led to the activation of Dual oxidase and resulted in increased midgut ROS levels. Our data indicate that the intestinal microbiota can be controlled in a blood-feeding vector by a novel, unprecedent mechanism, impacting also mosquito vectorial competence towards zika virus and possibly other pathogens as well. Author Summary Aminoacyl-tRNA synthetases (aaRS) are a family of ubiquitous enzymes responsible for the attachment of specific amino acids to their cognate tRNAs. During evolution some aaRS acquired new domains and/or suffered gene duplications, resulting in the improvement and expansion of their functions some of them being specific to a group of organisms. A paralog of seryl-tRNA synthetase restricted to the class Insecta (SLIMP) is found in Arthropoda. Our goal was to explore the role of SLIMP in the female mosquito Aedes aegypti using RNA interference. We showed that A. aegypti SLIMP (AaeSLIMP) gene expression is up-regulated upon blood feeding through a heme-dependent signaling. Although AaeSLIMP knockdown neither impacted the mosquito survival nor oviposition, it provoked ROS levels augmentation in the midgut via Dual Oxidase activity in order to control the increase in the intestinal native microbiota, specifically bacteria of the Bacteroidetes phylum. Although dysbiosis can result from mitochondrial impairment, this is the first time that the absence of a mitochondrial enzyme is linked to intestinal microbiota without any visible effects in mitochondrial respiration and mitochondrial ROS production. Furthermore, Zika Virus infection of AaeSLIMP silenced mosquitoes is decreased when comparing to control, meaning that Bacteroidetes overgrowth may be protecting the female mosquito. Our data indicate that the intestinal microbiota can be controlled in a blood-feeding vector by a novel, unprecedent mechanism, impacting also mosquito vectorial competence towards zika virus and possibly other pathogens as well.

Background: Declines in global malaria cases and deaths since the millennium are currently challenged by multiple factors including funding limitations, limits of, and resistance to vector control tools, and also recent spread of the invasive vector species, Anopheles stephensi – especially into novel urban settings where malaria rates are typically low. Coupled with general increases in urbanisation and escalations in the number of conflicts creating rapid and unplanned population displacement into urban areas, particularly in the Middle East and sub-Saharan Africa, increased urban malaria is a major threat to control and elimination. Methods: Entomological monitoring surveys (targeting Aedes aegypti ) of water containers across urban areas hosting internally displaced people (IDP) communities in Aden city, Yemen, were performed by The MENTOR Initiative, a non-governmental organisation. As part of these surveys, in 2021 In 23 larvae collected and raised to adults were morphologically identified as An. stephensi . Twelve of the samples were sent to Liverpool School of Tropical Medicine for independent morphological assessment and genetic analysis by sequencing the ribosomal ITS2 region and the mitochondrial COI gene. Results: All twelve samples were confirmed morphologically and by sequence comparisons to the NCBI BLAST database as An. stephensi , with phylogenetic analysis of the COI sequences identifying a single haplotype with closest relationship to a haplotype found in Djibouti and Ethiopia. Conclusion: Our results confirm the presence of An. stephensi in Yemen. Discovery of the species in an urban community hosting thousands of IDPs living in temporary shelters with widescale dependency on open water containers is of particular concern due to the vulnerability of the population and abundance of favourable breeding sites for the vector. Proactive monitoring and targeted integrated vector management are required to limit impacts in this area of typically low malaria transmission, and to prevent further the spread of An. stephensi within the region.

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

Evolution is a function of mutagenesis and selection. To analyse the role of mutagenesis on the structure of the SARS-CoV-2 genome, we reconstructed the mutational spectrum, which was highly C>U and G>U biased. This bias forces the SARS-CoV-2 genome to become increasingly U-rich unless selection cancels it. We analysed the consequences of this bias on the composition of the most neutral (four-fold degenerate synonymous substitutions) and the least neutral positions (nonsynonymous substitutions). The neutral nucleotide composition is already highly saturated by U and, according to our model, it is at equilibrium, suggesting that in the future, we don’t expect any more increase in U. However, nonsynonymous changes continue slowly evolve towards equilibrium substituting CG-rich amino-acids (“losers”) with U-rich ones (“gainers”). This process is universal for all genes of SARS-CoV-2 as well as for other coronaviridae species. In line with the direction mutation pressure hypothesis, we show that viral-specific amino acid content is associated with the viral-specific mutational spectrum due to the accumulation of effectively neutral slightly deleterious variants (losers to gainers) during the molecular evolution. The tuning of a protein space by the mutational process is expected to be typical for species with relaxed purifying selection, suggesting that the purging of slightly-deleterious variants in the SARS-CoV-2 population is not very effective, probably due to the fast expansion of the viral population during the pandemic. Understanding the mutational process can help to design more robust vaccines, based on gainer-rich motifs, close to the mutation-selection equilibrium.

Introduction: The impact of maternal coronavirus disease 2019 (COVID-19) infection on fetal health remains to be precisely characterized. Objectives: Using metabolomic profiling of newborn umbilical cord blood, we aimed to investigate the potential fetal biological consequences of maternal COVID-19 infection. Methods: : Cord blood serum samples from 23 mild COVID-19 cases (mother infected/ newborn negative) and 23 gestational age-matched controls were analyzed using nuclear magnetic spectroscopy and liquid chromatography coupled with mass spectrometry. Metabolite set enrichment analysis (MSEA) was used to evaluate altered biochemical pathways due to COVID-19 intrauterine exposure. Logistic regression models were developed using metabolites to predict intrauterine exposure. Results: : Significant concentration differences between groups (p-value <0.05) were observed in 19 metabolites. Elevated levels of glucocorticoids, pyruvate, lactate, purine metabolites, phenylalanine and branched chain amino acids of valine and isoleucine were discovered in cases while ceramide subclasses were decreased. The top metabolite model including cortisol and ceramide (d18:1/23:0) achieved an Area under the Receiver Operating Characteristics curve (95% CI) = 0.841 (0.725 - 0.957) for detecting fetal exposure to maternal COVID-19 infection. MSEA highlighted steroidogenesis, pyruvate metabolism, gluconeogenesis, and Warburg effect as the major perturbed metabolic pathways (p-value < 0.05). These changes indicate fetal increased oxidative metabolism, hyperinsulinemia, and inflammatory response. Conclusion: We present fetal biochemical changes related to intrauterine inflammation, altered energy metabolism in cases of mild maternal COVID-19 infection despite the absence of viral infection. Elucidation of the long-term consequences of these findings is imperative considering the large number of exposures in the population.

Three arboviruses, dengue virus, Zika virus and Japanese encephalitis virus, have wide distribution putting millions of people at risk of infection. These three flaviviruses show evolutionarily conserved features for the viral proteins, which consist of seven non-structural and three structural proteins. Non-structural protein 5 (NS5) is important for viral replication as it possesses multiple functions including both enzyme and non-enzyme roles. Oxidative stress induced by virus infection triggers glutathionylation of cell proteins. This study was to identify the effects of modification by glutathionylation on the guanylyltransferase activity of NS5 and identify the cysteine residues modified for the three flavivirus NS5 proteins. We found the three flavivirus proteins behaved in a similar fashion with increasing glutathionylation yielding decreasing guanylyltransferase activity. The three proteins also possessed conserved cysteines and these appeared to be modified for all three proteins. The glutathionylation appears to induce conformational changes that affect enzyme activity but possibly also create binding sites for host cell protein interactions that occur at later stages of viral propagation.

ABSTRACT RNA interference (RNAi) plays an essential role in mosquito antiviral immunity, but it is not known whether viral siRNA profiles differ between mosquito-borne and mosquito-specific viruses. A pan-Orthoflavivirus analysis in Aedes albopictus cells revealed that viral siRNAs were evenly distributed across the viral genome of most representatives of the Flavivirus genus. In contrast, siRNA production was biased towards the 3’ untranslated region (UTR) of the genomes of classical insect-specific flaviviruses (cISF), which was most pronounced for Kamiti River virus (KRV), a virus with a unique, 1.2 kb long 3’ UTR. KRV-derived siRNAs were produced in high quantities and almost exclusively mapped to the 3’ UTR. We mapped the 5’ end of KRV subgenomic flavivirus RNAs (sfRNAs), products of the 5’-3’ exoribonuclease XRN1/Pacman stalling on secondary RNA structures in the 3’ UTR of the viral genome. We found that KRV produces high copy numbers of a long, 1017 nt sfRNA1 and a short, 421 nt sfRNA2, corresponding to two predicted XRN1-resistant elements. Expression of both sfRNA1 and sfRNA2 was reduced in Pacman deficient Aedes albopictus cells, however, this did not correlate with a shift in viral siRNA profiles. We suggest that cISFs and particularly KRV developed a unique mechanism to produce high amounts of siRNAs as a decoy for the antiviral RNAi response in an sfRNA-independent manner. IMPORTANCE The Flavivirus genus contains diverse mosquito viruses ranging from insect-specific viruses circulating exclusively in mosquito populations to mosquito-borne viruses that cause disease in humans and animals. Studying the mechanisms of virus replication and antiviral immunity in mosquitoes is important to understand arbovirus transmission and may inform the development of disease control strategies. In insects, RNA interference (RNAi) provides broad antiviral activity and constitutes a major immune response against viruses. Comparing diverse members of the Flavivirus genus, we found that all flaviviruses are targeted by RNAi. However, the insect-specific Kamiti River virus was unique in that small interfering RNAs are highly skewed towards its uniquely long 3’ untranslated region. These results suggest that mosquito-specific viruses have evolved unique mechanisms for genome replication and immune evasion.

Abstract Background Patients infected with COVID-19 admitted to the intensive care unit may have a higher incidence of developing secondary infections. These infections can further deteriorate the hospital course and increase mortality. Therefore, the objectives of this study were to investigate the incidence, associated risk factors, outcomes, and pathogens associated with secondary bacterial infections in critically ill patients with COVID-19. Methods All adult COVID-19 patients admitted to the Intensive Care Unit requiring mechanical ventilation from 1st October 2020 until 31st December 2021 were screened for inclusion in the study. A total of 86 patients were screened, and 65 who met the inclusion criteria were prospectively entered into a customized electronic database. The database was then retrospectively analyzed to investigate secondary bacterial infections. 41.54% acquired at least one of the studied secondary bacterial infections during their ICU stay.. Results The most common secondary infection (59.26%) seen was hospital-acquired pneumonia followed by acquired bacteremia of unknown origin (25.92%), and catheter-related sepsis (14.81%). Diabetes mellitus (P = < 0.001), cumulative dose of corticosteroids (P = 0.001), and older age (P = < 0.001) were associated with an increased risk of secondary bacterial infection. The most commonly isolated pathogen in patients with secondary pneumonia was Acinetobacter baumannii. Staphylococcus aureus was the most common organism associated with a bloodstream infection or catheter-related sepsis. Conclusion Incidence of secondary bacterial/fungal infections was high in critically ill patients with COVID-19 and was associated with a longer duration of admission to the hospital and ICU and higher mortality. Age, a history of diabetes mellitus, and the administration of corticosteroids were associated with an increased risk of secondary bacterial infection.

Background Ongoing research of the mosquito microbiome aims to uncover novel strategies to reduce pathogen transmission. Sequencing costs, especially for metagenomics, are however still significant. A resource that is increasingly used to gain insights into host-associated microbiomes is the large amount of publicly available genomic data based on whole organisms like mosquitoes, which includes sequencing reads of the host-associated microbes and provides the opportunity to gain additional value of these initially host-focused sequencing projects. Methods To analyse non-host reads from existing genomic data, we developed a snakemake workflow called MINUUR ( M icrobial IN sights U sing U nmapped R eads). Within MINUUR, reads derived from the host-associated microbiome were extracted and characterised using taxonomic classifications and metagenome assembly followed by binning and quality assessment. We applied this pipeline to five publicly available Aedes aegypti genomic datasets, consisting of 62 samples with a broad range of sequencing depths. Results We demonstrate that MINUUR recovers previously identified phyla and genera and is able to extract bacterial metagenome assembled genomes (MAGs) associated to the microbiome. Of these MAGS, 42 are high-quality representatives with >90% completeness and <5% contamination. These MAGs improve the genomic representation of the mosquito microbiome and can be used to facilitate genomic investigation of key genes of interest. Furthermore, we show that samples with a high number of KRAKEN2 assigned reads produce more MAGs. Conclusions Our metagenomics workflow, MINUUR, was applied to a range of Aedes aegypti genomic samples to characterise microbiome-associated reads. We confirm the presence of key mosquito-associated symbionts that have previously been identified in other studies and recovered high-quality bacterial MAGs. In addition, MINUUR and its associated documentation are freely available on GitHub ( https://github.com/aidanfoo96/MINUUR ) and provide researchers with a convenient workflow to investigate microbiome data included in the sequencing data for any applicable host genome of interest.

Aedes aegypti mosquitoes can transmit several arboviruses, including chikungunya virus (CHIKV), dengue virus (DENV), and Zika virus (ZIKV). When blood-feeding on a virus-infected human, the mosquito ingests the virus into the midgut (stomach), where it replicates and must overcome the midgut barrier to disseminate to other organs and ultimately be transmitted via the saliva. Current tools to study mosquito-borne viruses (MBVs) include 2D-cell culture systems and in vivo mosquito infection models, which offer great advantages, yet have some limitations. Here, we describe a long-term ex vivo culture of Ae. aegypti midguts. Cultured midguts were metabolically active for 7 days in a 96-well plate at 28°C and were permissive to ZIKV, DENV, Ross River virus (RRV) and CHIKV. Ex vivo midguts from Culex pipiens mosquitoes were found to be permissive to Usutu virus (USUV). Immunofluorescence staining confirmed viral protein synthesis in CHIKV-infected midguts of Ae. aegypti . Furthermore, fluorescence microscopy revealed replication and spread of a reporter DENV in specific regions of the midgut. In addition, two known antiviral molecules, β-D-N 4 -hydroxycytidine (NHC) and 7-deaza-2’- C -methyladenosine (7DMA), were able to inhibit CHIKV and ZIKV replication, respectively, in the ex vivo model. Together, our results show that ex vivo midguts can be efficiently infected with mosquito-borne alpha- and flaviviruses and employed to evaluate antiviral drugs. Furthermore, the setup can be extended to other mosquito species. Ex vivo midgut cultures could thus be a new model to study MBVs, offering the advantage of reduced biosafety measures compared to infecting living mosquitoes. Importance Mosquito-borne viruses (MBVs) are a significant global health threat since they can cause severe diseases in humans, such as hemorrhagic fever, encephalitis, and chronic arthritis. MBVs rely on the mosquito vector to infect new hosts and perpetuate virus transmission. No therapeutics are currently available. The study of arbovirus infection in the mosquito vector can greatly contribute to elucidating strategies for controlling arbovirus transmission. This work investigated the infection of midguts from Aedes aegypti mosquitoes in an ex vivo platform. We found several MBVs capable of replicating in the midgut tissue, including viruses of major health importance, such as dengue, chikungunya, and Zika viruses. Additionally, antiviral compounds reduced arbovirus infection in the cultured midgut tissue. Overall, the midgut model emerges as a useful tool for diverse applications such as studying tissue-specific responses to virus infection and screening potential anti-arboviral molecules.

Summary CD4 + T follicular helper (T FH ) cells are required for high-quality antibody generation and maintenance. However, the longevity and functional role of these cells are poorly defined in COVID-19 convalescents and vaccine recipients. Here, we longitudinally investigated the dynamics and functional roles of spike-specific circulating T FH cells and their subsets in convalescents at the 2 nd , 5 th , 8 th , 12 th and 24 th months after COVID-19 symptom onset and in vaccinees after two and three doses of inactivated vaccine. SARS-CoV-2 infection elicited robust spike-specific T FH cell and antibody responses, of which spike-specific CXCR3 + T FH cells but not spike-specific CXCR3 − T FH cells and neutralizing antibodies were persistent for at least two years in more than 80% of convalescents who experienced symptomatic COVID-19, which was well coordinated between spike-specific T FH cell and antibody responses at the 5 th month after infection. Inactivated vaccine immunization also induced spike-specific T FH cell and antibody responses; however, these responses rapidly declined after six months with a two-dose standard administration, and a third dose significantly promoted antibody maturation and potency. Functionally, spike-specific CXCR3 + T FH cells exhibited better responsiveness than spike-specific CXCR3 − T FH cells upon spike protein stimulation in vitro and showed superior capacity in supporting spike-specific antibody secreting cell (ASC) differentiation and antibody production than spike-specific CXCR3 − T FH cells cocultured with autologous memory B cells. In conclusion, spike-specific CXCR3 + T FH cells played a dominant functional role in antibody elicitation and maintenance in SARS-CoV-2 infection and vaccination, suggesting that induction of CXCR3-biased spike-specific T FH cell differentiation will benefit SARS-CoV-2 vaccine development aiming to induce long-term protective immune memory. Highlights SARS-CoV-2 infection elicited robust spike-specific T FH cell and antibody responses, which persisted for at least two years in the majority of symptomatic COVID-19 convalescent patients. Inactivated vaccine immunization also elicited spike-specific T FH cell and antibody responses, which rapidly declined over time, and a third dose significantly promoted antibody maturation and potency. Spike-specific CXCR3 + T FH cells exhibited more durable responses than spike-specific CXCR3 − T FH cells, correlated with antibody responses and showed superior capacity in supporting ASC differentiation and antibody production than spike-specific CXCR3 − T FH cells.

The primary route of Zika virus (ZIKV) transmission is through the bite of an infected Aedes mosquito, when it probes the skin of a vertebrate host during a blood meal. Viral particles are injected into the bite site together with mosquito saliva and a complex mixture of other components. Some of them are shown to play a key role in the augmentation of the arbovirus infection in the host, with increased viremia and/or morbidity. This vector-derived contribution to the infection is not usually considered when vaccine candidates are tested in preclinical animal models. In this study, we performed a preclinical validation of a promising ZIKV vaccine candidate in a mosquito-mouse transmission model using both Asian and African ZIKV lineages. Mice were immunized with engineered ZIKV virus-like particles and subsequently infected through the bite of ZIKV-infected Ae. aegypti mosquitoes. Despite a mild increase in viremia in mosquito-infected mice compared to those infected through traditional needle injection, the vaccine protected the animals from developing the disease and strongly reduced viremia. In addition, during peak viremia, naïve mosquitoes were allowed to feed on infected vaccinated and non-vaccinated mice. Our analysis of viral titers in mosquitos showed that the vaccine was able to inhibit virus transmission from the host to the vector. Author summary Zika is a mosquito-borne viral disease, causing acute debilitating symptoms and complications in infected individuals and irreversible neuronal abnormalities in newborn children. The primary vectors of ZIKV are generally considered to be mosquitoes of the genus Aedes , in particular Aedes aegypti . Despite representing a significant public health burden with a widespread transmission in many regions of the world, Zika remains a neglected disease with no effective antiviral therapies or approved vaccines to control and prevent infections. The efficacy of several promising candidate vaccines is however under investigation, mainly through artificial infections (i.e. needle-mediated injections of the virus) in animal models, while it is known that components of the mosquito bite lead to an enhancement of viral infection and spread. In this study, we have also included mosquitoes as viral vectors, demonstrating that the ability of a promising candidate vaccine to protect animals against ZIKV infections after the bite of an infected mosquito, and to also prevent its further transmission. These findings represent an additional crucial step for the development of an effective prevention tool for clinical use. Graphical abstract

Non-coding RNA structure and function are essential to understanding various biological processes, such as cell signaling, gene expression, and post-transcriptional regulations. These are all among the core problems in the RNA field. With the rapid growth of sequencing technology, we have accumulated a massive amount of unannotated RNA sequences. On the other hand, expensive experimental observatory results in only limited numbers of annotated data and 3D structures. Hence, it is still challenging to design computational methods for predicting their structures and functions. The lack of annotated data and systematic study causes inferior performance. To resolve the issue, we propose a novel RNA foundation model (RNA-FM) to take advantage of all the 23 million non-coding RNA sequences through self-supervised learning. Within this approach, we discover that the pre-trained RNA-FM could infer sequential and evolutionary information of non-coding RNAs without using any labels. Furthermore, we demonstrate RNA-FM's effectiveness by applying it to the downstream secondary/3D structure prediction, SARS-CoV-2 genome structure and evolution prediction, protein-RNA binding preference modeling, and gene expression regulation modeling. The comprehensive experiments show that the proposed method improves the RNA structural and functional modelling results significantly and consistently. Despite only being trained with unlabelled data, RNA-FM can serve as the foundational model for the field.

Background Exposure to elevated interleukin (IL)-6 levels in utero is consistently associated with increased risk for psychiatric disorders with a putative neurodevelopmental origin, such as schizophrenia (SZ) and autism spectrum condition (ASC). Although rodent models provide causal evidence for this association, we lack a detailed understanding of the cellular and molecular mechanisms in human model systems. To close this gap, we characterised the response of hiPSC-derived microglia-like cells (MGL) and neural progenitor cells (NPCs) to IL-6 in monoculture. Results We observed that human forebrain NPCs did not respond to acute IL-6 exposure in monoculture at both a protein and transcript level due to the absence of IL-6Ra expression and sIL-6Ra secretion. By contrast, acute IL-6 exposure resulted in STAT3 phosphorylation and increased IL-6, JMJD3 and IL-10 expression in MGL, confirming activation of canonical IL-6R signalling. Bulk RNAseq identified 156 upregulated genes (FDR <0.05) in MGL following acute IL-6 exposure, including IRF8, REL, HSPA1A/B and OXTR , which significantly overlapped with an upregulated gene set from post-mortem brain tissue from individuals with schizophrenia. Acute IL-6 stimulation significantly increased MGL motility suggestive of a gain of surveillance function, consistent with gene ontology pathways highlighted from the RNAseq data. Finally, MGLs displayed elevated CCL1, CXCL1, MIP-1A/B, IL-8, IL-13, IL-16, IL-18, MIF and Serpin-E1 secretion post 3h and 24h IL-6 exposure. Conclusion Our data provide evidence for cell specific effects of acute IL-6 exposure in a human model system and strongly suggest microglia-NPC co-culture models are required to study how IL-6 influences human cortical neural progenitor cell development in vitro .

Chikungunya virus (CHIKV) is an Aedes mosquito-borne virus that has caused explosive epidemics linked to acute, chronic, and severe clinical outcomes. Since 2014, Brazil has had the highest number of chikungunya fever (CHIKF) cases in the Americas. Here, we report and contextualize the spatiotemporal dynamic of CHIKF in Brazil and combine genomic, epidemiological, and vector analyses to investigate CHIKF recurrence in several Brazilian states. From 2013 to 2022, CHIKV caused seven epidemic waves across Brazil, affecting 59.5% (3,316 of 5,570) of the country’s municipalities. To date, Ceará State in the northeast has been the most affected, with 81,274 cases during the two largest epidemic waves in 2016 and 2017, and the ongoing third wave in 2022. The 2022 CHIKF recurrence was associated with a new introduction of an East/Central/South African strain. Also, the CHIKV recurrences in Ceará, Tocantins, and Pernambuco States were limited to municipalities with few or no prior reported cases in the previous epidemic waves, suggesting that spatial heterogeneity of CHIKV spread and population immunity may explain the recurrence pattern in the country. In addition, the population density metrics of main CHIKV vector in Brazil, Ae. aegypti , were not correlated spatially with locations of CHIKF recurrence in Ceará and Tocantins States. Also, we show that CHIKF disproportionally affected females, and we estimated the case-fatality ratio in Ceará at ∼1.3 deaths per 1,000 cases. These findings more comprehensively describe CHIKV epidemics in Brazil and contribute to understanding CHIKF recurrence in urban settings. Overall, this information may help guide public health policy to mitigate and reduce the burden of urban arboviruses.