The consequences of Zika virus (ZIKV) infections were limited to sporadic mild diseases until almost a decade ago, when epidemics outbreaks took place and were followed by virus introduction and quick spread into the Americas. Simultaneously, novel severe neurological manifestations for ZIKV infections were identified, including congenital microcephaly. However, the complete understanding of why the epidemic strains behave differently from previously circulating variants is still missed and many questions still remain about the actual significance of ZIKV genetic variations in the epidemiology and biology of ZIKV. Here, we attained to analyze a high number of sequences to identify genes with different levels of variability and patterns of genomic variations that could be associated with ZIKV diversity. We compared numerous epidemic strains with the pre-epidemic ones from both the Asian and the African lineages, while we also investigated special variations among the epidemic ZIKV strains derived from microcephaly cases. We identified several viral genes with dissimilar mutation rates among the ZIKV strain groups. Moreover, we found novel protein variation profiles that might be associated with the epidemiological particularities of each category of ZIKV strains. Finally, we assessed the impact of the detected changes on the structure and stability of the corresponding viral proteins, finding some interesting non synonymous variations which could help to explain the heterogeneous features among the diverse ZIKA strains. Altogether, this work contributes to identify ZIKV genetic variations that might have a phenotypical impact, as a basis for future experimental analysis to elucidate the recent ZIKV emergency.

Summary Zika virus (ZIKV) is an arthopod-vectored flavivirus that disseminates from the infection site into peripheral tissues, where it can elicit virus-induced pathology. To move through the body, ZIKV is thought to exploit the mobility of myeloid cells, in particular monocytes and dendritic cells. However, multiple distinct steps during viral spread culminate in peripheral tissue infection, and the timing and mechanisms underlying mobile immune cell shuttling of virus remain unclear. To understand the very early steps in ZIKV dissemination from the skin, we kinetically and spatially mapped ZIKV-infected lymph nodes (LNs), an intermediary stop en route to the blood. Contrary to dogma, migratory immune cells were not required for large quantities of virus to reach the LN or blood. Instead, ZIKV rapidly infected a subset of immobile macrophages in the LN, which shed virus through the lymphatic pathway into the blood. Importantly, infection of LN macrophages alone was sufficient to initiate viremia. Together, our studies indicate that sessile macrophages that live and die in the LN contribute to initial ZIKV spread to the blood. These data build a more complete picture of ZIKV movement through the body and identify an alternate anatomical site for potential antiviral intervention. Highlights ZIKV infects and replicates in distinct LN macrophage populations LN macrophage infection results in infectious virus in the blood Virus reaches the blood in the absence of DC migration or monocyte infection Nodal macrophage infection does not sustain viremia or induce morbidity Graphical abstract Caption: ZIKV infects lymph node macrophages, which shed infectious virus into the lymph and then blood.

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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.

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.

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.

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.

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 Flaviviruses subvert the host RNA decay machinery to produce subgenomic flavivirus RNA (sfRNA), products of the 5’-3’ exoribonuclease XRN1/Pacman stalling on secondary RNA structures in the 3’ untranslated region (UTR) of the viral genome. The classical insect-specific flavivirus (cISF) Kamiti River virus (KRV) has a unique 1.2 kb long 3’ UTR, of which only 40% is homologous to its closest family member, cell-fusing agent virus (CFAV). We mapped the 5’ end of KRV sfRNAs and 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. Furthermore, we identified a new positive and negative sense 1.5 kb subgenomic RNA species that is colinear with the 3’ region of the NS5 gene and the viral 3’ UTR, which we tentatively named subgenomic cISF RNA (cifRNA). Expression of both sfRNA1 and sfRNA2 was reduced in Pacman deficient Aedes albopictus mosquito cells, while expression of the longer cifRNA was Pacman -independent. Interestingly, a pan-Flavivirus small RNA analysis in Aedes albopictus cells revealed that nearly all KRV-derived siRNAs mapped to the 3’ UTR region and that these siRNAs are produced in high quantity. 3’ UTR-biased siRNA production appeared to be conserved in other cISFs, albeit to a lesser extent, whereas siRNAs were evenly distributed across the viral genome of other representatives of the Flavivirus genus. We suggest that cISFs and particularly KRV developed a unique mechanism to produce high amounts of siRNA as a decoy of the antiviral RNAi response. 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, constituting the main immune response against viruses. Comparing the RNAi response across 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. Moreover, we found that Kamiti River virus produces a new subgenomic RNA species in addition to subgenomic flavivirus RNAs previously observed for other flaviviruses. These results suggest that mosquito-specific viruses have evolved unique mechanisms for genome replication and immune evasion.

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.

1 The microbiome is a collection of microbes that exist in symbiosis with a host. Whole genome sequencing produces off-target, non-specific reads, to the host in question, which can be used for metagenomic inference of a microbiome. This data is advantageous over barcoding methods since higher taxonomic resolution and functional predictions of microbes are possible. With the growing number of genomic sequencing data publicly available, comes opportunity to elucidate reads pertaining to the microbiome. However, characterization of these reads can be complex, with many steps required to perform a robust analysis. To address this, we developed MINUUR ( M icrobial IN sights U sing U nmapped R eads); a snakemake pipeline to characterize non-host reads from existing genomic data. We apply this pipeline to ten, publicly available, high coverage Aedes aegypti ( Ae. aegypti ) genomic samples. Using MINUUR, we describe species level microbial classifications; predict microbe associated genes and pathways and find bacterial metagenome assembled genomes (MAGs) associated to the Ae. aegypti microbiome. Of these MAGS, 19 are high-quality representatives with over 90% completeness and under 5% contamination. In summary, we present an in-depth analysis of non-host reads from Ae. aegypti whole genome sequencing data within a reproducible and open-access pipeline.

Aedes aegypti mosquitoes are known vectors of several arboviruses, including the 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 must replicate and overcome the midgut barrier to disseminate to other organs (e.g., salivary glands) and ultimately be transmitted via the saliva. Tools to study mosquito-borne viruses (MBVs) include two-dimensional cell culture systems and in vivo mosquito infection models, which offer great advantages, yet have some limitations. Here, we describe for the first time a long-term ex vivo culture of Ae. aegypti midguts. Cultured midguts were viable for 7 days in a 96-well plate at 28°C and displayed peristaltic movements. Several representative MBVs efficiently replicated in the midguts: ZIKV, DENV, Ross River virus (RRV) and CHIKV, as measured by qRT-PCR and plaque assay. Fluorescence microscopy revealed replication and spread of a fluorescent reporter virus (DV2/mCherry) in the midgut. Finally, we evaluated the antiviral activity of the nucleoside analogs β-D-N 4 -hydroxycytidine (NHC) and 7-deaza-2’- C -methyladenosine (7DMA), in the ex vivo midgut model. At 50 µM, NHC reduced the infectious virus levels at day 3 post-infection (p.i.) for both RRV and CHIKV by 4.5- and 9.4-fold, respectively. Likewise, 7DMA at 100 µM reduced ZIKV RNA viral loads by 20-fold at day 7 p.i..Together, our results show that ex vivo midguts can be efficiently infected with mosquito-borne alpha- and flaviviruses. Furthermore, the cultured midguts could potentially be used to evaluate the antiviral activity of inhibitors. 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.

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 .

Background: The WHO Unity Studies initiative supports countries, especially low- and middle-income countries (LMICs), to conduct seroepidemiologic studies for rapidly informing responses to the COVID-19 pandemic. Ten generic study protocols were developed which standardized epidemiologic and laboratory methods. WHO provided technical support, serological assays, and funding for study implementation. An external evaluation was conducted to assess 1) the usefulness of study findings in guiding response strategies, 2) management and support to conduct studies, and 3) capacity built from engagement with the initiative. Methods: . The evaluation focused on the three most used protocols, first few cases, household transmission, and population-based serosurvey, 66% of 339 studies tracked by WHO. All 158 principal investigators (PIs) with contact information were surveyed online. Interviews were conducted with 15 PIs (randomly selected within WHO regions), 14 WHO Unity focal points at country, regional and global levels, 12 WHO global-level stakeholders, and eight external partners. Interviews were coded in MAXQDA™, synthesized into findings, and cross-verified by a second reviewer. Results: . Among 69 (44%) survey respondents, 61 (88%) were from LMICs. Ninety-five percent gave positive feedback on technical support, 87% reported findings contributed to COVID-19 understanding, 65% for guiding public health and social measures, and 58% for guiding vaccination policy. Survey and interview groups responses showed that the main technical barriers to using study findings were study quality, variations in study methods (challenge for meta-analysis), completeness of reporting study details, and clarity of communicating findings. Untimely study findings was another barrier, caused by delays in ethical clearance, receipt of serological assays, and approval to share findings. There was strong agreement that the initiative created equitable research opportunities, connected expertise, and facilitated study implementation. Around 90% of respondents agreed the initiative should continue in the future. Conclusions: . The Unity Studies initiative created a highly valued community of practice, contributed to study implementation and research equity, and serves as valuable framework for future pandemics. To strengthen this platform, WHO should establish emergency-mode procedures to facilitate timeliness and continue to build capacity to rapidly conduct high-quality studies and communicate findings in a format friendly to decision-makers.

ABSTRACT Aedes aegypti vectors the pathogens that cause dengue, yellow fever, Zika virus, and chikungunya, and is a serious threat to public health in tropical regions. Decades of work has illuminated many aspects of Ae. aegypti ’s biology and global population structure, and has identified insecticide resistance genes; however, the size and repetitive nature of the Ae. aegypti genome have limited our ability to detect positive selection in this mosquito. Combining new whole genome sequences from Colombia with publicly available data from Africa and the Americas, we identify multiple strong candidate selective sweeps in Ae. aegypti , many of which overlap genes linked to or implicated in insecticide resistance. We examine the voltage-gated sodium channel gene in three American cohorts, and find evidence for successive selective sweeps in Colombia. The most recent sweep encompasses an intermediate-frequency haplotype containing four candidate insecticide resistance mutations that are in near-perfect linkage disequilibrium with one another in the Colombian sample. We hypothesize that this haplotype may continue to rapidly increase in frequency and perhaps spread geographically in the coming years. These results extend our knowledge of how insecticide resistance has evolved in this species, and add to a growing body of evidence suggesting Ae. aegypti has an extensive genomic capacity to rapidly adapt to insecticide-based vector control.

The mosquito Aedes aegypti is the vector of a number of medically-important viruses, including dengue virus, yellow virus, chikungunya virus, and Zika virus, and as such vector control is a key approach to managing the diseases they cause. Understanding the impact of vector control on these diseases is aided by first understanding its impact on Ae. aegypti population dynamics. A number of detail-rich models have been developed to couple the dynamics of the immature and adult stages of Ae. aegypti . The numerous assumptions of these models enable them to realistically characterize impacts of mosquito control, but they also constrain the ability of such models to reproduce empirical patterns that do not conform to the models’ behavior. In contrast, statistical models afford sufficient flexibility to extract nuanced signals from noisy data, yet they have limited ability to make predictions about impacts of mosquito control on disease caused by pathogens that the mosquitoes transmit without extensive data on mosquitoes and disease. Here, we demonstrate how the differing strengths of mechanistic realism and statistical flexibility can be fused into a single model. Our analysis utilizes data from 176,352 household-level Ae. aegypti aspirator collections conducted during 1999-2011 in Iquitos, Peru. The key step in our approach is to calibrate a single parameter of the model to spatio-temporal abundance patterns predicted by a generalized additive model (GAM). In effect, this calibrated parameter absorbs residual variation in the abundance time-series not captured by other features of the mechanistic model. We then used this calibrated parameter and the literature-derived parameters in the agent-based model to explore Ae. aegypti population dynamics and the impact of insecticide spraying to kill adult mosquitoes. The baseline abundance predicted by the agent-based model closely matched that predicted by the GAM. Following spraying, the agent-based model predicted that mosquito abundance rebounds within about two months, commensurate with recent experimental data from Iquitos. Our approach was able to accurately reproduce abundance patterns in Iquitos and produce a realistic response to adulticide spraying, while retaining sufficient flexibility to be applied across a range of settings. Author Summary The mosquito Aedes aegypti is the vector for a number of the most medically important viruses, including dengue, Zika, chikungunya, and yellow fever. Understanding the population dynamics of this mosquito, and how those dynamics might respond to vector control interventions, is critical to inform the deployment of such interventions. One of the best ways to gain this understanding is through modeling of population dynamics. Such models are often categorizes as either statistical or dynamical, and each of these approaches has advantages and disadvantages – for instance, statistical models may more closely match patterns observed in empirical data, while dynamical models are better able to predict the impact of counterfactual situations such as vector control strategies. In this paper, we present an approach which fuses these two approaches in order to gain the advantages of both: it fits empirical data on Aedes aegypti population dynamics well, while producing realistic responses to vector control interventions. Our approach has the potential to inform and improve the deployment of vector control interventions, and, when used in concert with and epidemiological model, to help reduce the burden of the diseases spread by such vectors.