Aedes albopictus is a competent vector of several arboviruses that has spread throughout the United States over the last three decades after it was initially detected in Texas in 1985. With the emergence of Zika virus in the Americas in 2015-2016 and an increased need to better understand the current distributions of Ae. albopictus in the US, we initiated surveillance efforts to determine the abundance of invasive Aedes species in Iowa. Here, we describe the resulting surveillance efforts from 2016-2020 in which we detect stable and persistent populations of Aedes albopictus in three Iowa counties. Based on temporal patterns in abundance and genetic analysis of mitochondrial DNA haplotypes between years, our data support that populations of Ae. albopictus are overwintering and have likely become established in the state. In addition, the localization of Ae. albopictus predominantly in areas of urbanization and noticeable absence in rural areas suggests that these ecological factors may represent potential barriers to their further spread and contribute to overwintering success. Together, these data document the establishment of Ae. albopictus in Iowa and their expansion into the Upper Midwest, where freezing winter temperatures were previously believed to limit their spread. With increasing globalization, urbanization, and rising temperatures associated with global warming, the range of invasive arthropod vectors, such as Ae. albopictus, is expected to only further expand, creating increased risks for vector-borne disease.

Zika virus (ZIKV) infection at the maternal-placental interface is associated with adverse pregnancy outcomes including fetal demise and pregnancy loss. To determine how infection impacts placental trophoblasts, we utilized rhesus macaque trophoblast stem cells (TSC) that can be differentiated into early gestation syncytiotrophoblasts (ST) and extravillous trophoblasts (EVT). TSCs and STs, but not EVTs, were highly permissive to productive infection with ZIKV strain DAK AR 41524. The impact of ZIKV on the cellular transcriptome showed that infection of TSCs and STs increased expression of immune related genes, including those involved in type I and type III interferon responses. ZIKV exposure altered extracellular vesicle (EV) protein, mRNA, and miRNA cargo, regardless of productive infection. These findings suggest that early gestation macaque TSCs and STs are permissive to ZIKV infection, and that EV analysis may provide a foundation for identifying non-invasive biomarkers of placental infection in a highly translational model.

The ability of viruses to evade the host antiviral immune system determines their level of replication fitness, species specificity, and pathogenic potential. Flaviviruses rely on the subversion of innate immune barriers including the type I and type III IFN antiviral systems. Zika virus infection induces the degradation of STAT2, an essential component of the IFN stimulated gene transcription factor, ISGF3. The mechanisms that lead to STAT2 degradation by Zika virus are poorly understood, but it is known to be mediated by the viral NS5 protein that binds to STAT2 and targets it for proteasome-mediated destruction. To better understand how NS5 engages and degrades STAT2, functional analysis of the protein interactions that lead to Zika virus and NS5-dependent STAT2 proteolysis were investigated. Data implicate the STAT2 coiled-coil domain as necessary and sufficient for NS5 interaction and proteasome degradation after Zika virus infection. Molecular dissection reveals that the first two α-helices of the STAT2 coiled-coil contain a specific targeting region for IFN antagonism. These functional interactions provide a more complete understanding of the essential protein-protein interactions needed for Zika virus evasion of the host antiviral response, and identifies new targets for antiviral therapeutic approaches. Importance Zika virus infection can cause mild fever, rash, and muscle pain, and in rare cases lead to brain or nervous system diseases including Guillain–Barré syndrome. Infections in pregnant women can increase the risk of miscarriage or serious birth defects including brain anomalies and microcephaly. There are no drugs or vaccines for Zika disease. Zika virus is known to break down the host antiviral immune response, and this research project reveals how the virus suppresses interferon signaling, and may reveal therapeutic vulnerabilities.

Neonatal microcephaly and adult Guillain-Barré syndrome are severe complications of Zika virus (ZIKV) infection. The robustly induced inflammatory cytokine expressions in ZIKV-infected patients may constitute a hallmark for severe disease. In the present study, the potential role of high mobility group box 1 protein (HMGB1) in ZIKV infection was investigated. HMGB1 protein expression was determined by the enzyme-linked immunosorbent assay (ELISA) and immunoblot assay. HMGB1’s role in ZIKV infection was also explored using treatment with dexamethasone, an immunomodulatory drug. Antiviral effects of dexamethasone treatment on both wild-typed (WT) and HMGB1-knockdown (shHMGB1) Huh7 cells were determined by the focus-forming assay. Results showed that the Huh7 cells were highly susceptible to ZIKV infection. The infection was found to induce HMGB1 nuclear-to-cytoplasmic translocation, resulting in a >99% increase in the cytosolic HMGB1 expression at 72h.p.i. The extracellular HMGB1 level was elevated in a time- and multiplicity of infection (MOI)- dependent manner. Dexamethasone 150 µM treatment of the ZIKV-infected cells reduced HMGB1 extracellular release in a dose-dependent manner, with a maximum reduction of 71 ± 5.84% (p < 0.01). The treatment also reduced virus titers by over 83 ± 0.50% (p < 0.01). The antiviral effects, however, was not observed in the dexamethasone-treated HMGB1-knockdown cells, suggesting the importance of the intracellular HMGB1 in ZIKV infection. Overall, these results suggest that translocation of HMGB1 occurred during ZIKV infection and inhibition of the translocation reduced ZIKV replication. These findings highlight the potential of developing therapeutics against ZIKV infection by affecting the translocation of HMGB1 from the nucleus to the cytoplasm.

Zika virus (ZIKV) emergence highlighted the need for a deeper understanding on virus-host interaction to pave the development of antiviral therapies. The present work aimed to address the response of neutrophils during ZIKV infection. Neutrophils are an important effector cell in innate immunity involved in the host response to neurotropic arboviruses. Our results indicate that human neutrophils were not permissive to Asian or African ZIKV strains replication. Indeed, after stimulation with ZIKV, neutrophils were not primed against the virus as evaluated by the absence of CD11b modulation, secretion of inflammatory cytokines and granule content, production of reactive oxygen species and neutrophil extracellular traps formation. Overall, neutrophils did not affect ZIKV infectivity. Moreover, ZIKV infection of primary innate immune cells in vitro did not trigger neutrophil migration. However, neutrophil co-cultured with ZIKV susceptible cells (A549) resulted in lower frequencies of infection on A549 cells by cell-to-cell contact. In vivo , neutrophil depletion from immunocompetent mice did not affect ZIKV spreading to the draining lymph nodes. The data suggest human neutrophils do not play a per se antiviral role against ZIKV, but these cells might participate in an infected environment shaping the ZIKV infection in other target cells.

Zika virus (ZIKV) can be transmitted from mother to fetus during pregnancy, causing adverse fetal outcomes. Several studies have indicated that ZIKV can damage the fetal brain directly; however, whether the ZIKV-induced maternal placental injury contribute to adverse fetal outcomes are sparsely defined. Here, we discovered that ZIKV causes the pyroptosis of placental cells by activating the executor Gasdermin E (GSDME) in vitro and in vivo . Mechanistically, caspase-8 undergoes activation upon the recognition of 5’ untranslated region of viral RNA by RIG-I, followed by the stimulation of caspase-3 to ultimately escalate the GSDME cleavage. Further analyses revealed that the ablation of GSDME in ZIKV-infected pregnant mice attenuates placental pyroptosis, which consequently confers protection against adverse fetal outcomes. In conclusion, our study unveils a novel mechanism of ZIKV-induced adverse fetal outcomes via causing placental cell pyroptosis, which could be employed for developing new therapies for ZIKV-associated diseases. Significance statement Several studies have elucidated the link between ZIKV infection and congenital ZIKV syndroms (CZS), but the pathogenesis yet needs further study. Here, we reported a novel pathogenic mechanism of ZIKV which leads to pyroptosis of placental cells through activating the pyroptotic executor GSDME, rather than GSDMD. Upon ZIKV infection, GSDME-mediated pyroptosis damages the structure and function of the placenta, thereby affecting the development of the fetus and contributing to the adverse fetal outcomes. Our study highlights the importance of pyroptotic executor GSDME in regulate ZIKV pathogenicity and further confirms that placental injury caused by ZIKV infection is a key factor for CZS.

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular method for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, ‘SMART-9N’, and a version compatible with barcoded PCR primers available from Oxford Nanopore Technologies, ‘Rapid SMART-9N’, for the detection, characterization, and whole-genome sequencing of RNA viruses. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6e00 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

ABSTRACT ZIKV is a 11Kb positive stranded flavivirus transmitted by infected Aedes aegypti and by sexual intercourse. After a short period of viremia of 5-7 days, the virus is cleared, and infection resolved in 80% of individuals. However, around 27% of the fetuses from pregnant infected mothers may develop several fetal brain and ocular pathology. Here we show that murine and peripheral blood human neutrophils support ZIKV infection and replication both in vitro and in vivo , which may correlate to the facilitation of vertical transmission. ZIKV did not interfere with cell viability, neither induced ROS production nor the release of NETs by infected neutrophils. Also, ZIKV infection of neutrophils did not trigger a pro-inflammatory profile, as evidenced by qPCR and proteomic analysis. Interestingly, ZIKV-infected neutrophils were isolated from the placenta were highly infected. The transference of in vitro ZIKV-infected neutrophils to pregnant female mice favored the transference of viral particles to the fetus. Conversely, neutrophil depletion with monoclonal antibodies reduced fetal viral loads whereas the treatment with recombinant G-CSF has the opposite effect. In summary, although it has already been shown that circulating monocytes harbor ZIKV, to our knowledge, this is the first report demonstrating the role of neutrophils during ZIKV infection, and most important, that it may act as a trojan horse to placental tissue directly impacting the pathogenesis of congenital syndrome.

Background: The invasive species Aedes albopictus , the Asian tiger mosquito, has undergone an extreme expansion by steady introductions as blind passengers in vehicles from the Mediterranean to South-West Germany. The more than 15 established populations in the State of Baden-Württemberg and Palatine (South-West Germany) have become a major nuisance and public health threat. Aedes albopictus deserves special attention as vector of arboviruses like dengue, chikungunya or Zika virus. In Germany, control of Ae. albopictus is implemented under the auspice of health departments and regulatory offices. Methods: The control strategy comprised three components or pillars: a) community participation (CP) based on the elimination or sanitation of breeding sites with the use of fizzy Bti-tablets (Culinex Tab plus); b) Door-to-Door (DtD) control by trained staff applying high doses of a Bti-water-dispersible granular formulation (Vectobac WG) aimed for a long-lasting killing effect; and c) the Sterile Insect Technique (SIT) to eliminate remaining Ae. albopictus populations. Prior to large scale routine city-wide treatments, the efficacy of the three elements was evaluated in laboratory and semi-field trials. Special emphasis was given to the mass release of Ae. albopictus sterile males. Results: More than 60% of the local residents joined the Community Participation within the large-scale control program. It was shown that the most effective element was the DtD intervention including the application of Vectobac WG (2700 ITU/mg after radiation with 25 kGy) to potential breeding sites (10 g/rainwater container, max. 200L ≙ 13,500ITU/L and 2.5g/container <50L) with a persistence of at least about three weeks. The average time required for the inspection and treatment per property was 27 minutes. In Ludwigshafen the larval source management resulted in a container index for Ae. albopictus below 1% in 2020 compared to 10.9% in 2019. The mean number of Aedes eggs/ovitrap were 4.3 in Ludwigshafen and 18.23 in Freiburg-Metzgergrün (SIT areas); while 22.4 in Freiburg-Gartenstadt (Control area). After the strong reduction of the Aedes population by Bti-application, the weekly release of 1,013 (Ludwigshafen) and 2,320 (Freiburg) sterile Ae. albopictus males/ha from May until October resulted in a high percentage of sterile eggs. In the trial area of Ludwigshafen the sterility of eggs reached 82.61% (mean: 60.52%; SD: 42.88%) and in Freiburg 62.68% (SD 28.21%). The natural sterility in the control area was 16.93±13.5%. The field results were in line with data obtained in cage tests under laboratory conditions where wild females mated with sterile males showed sterility rates of 87.53±9.15%. The sterility of eggs laid by females mated with unirradiated males was only 3.3±2.8%. The overall sterility of about 83% in Ludwigshafen indicates that our goal to almost eradicate the Ae. albopictus population could be achieved. Conclusions: It is shown that an integrated control program based on a strict monitoring scheme is most effective when it comprises three components, namely a) community participation, b) DtD intervention including long-lasting Bti-larviciding to strongly reduce Ae. albopictus populations and c) the release of sterile males to reduce the remaining Ae. albopictus population to a minimum or even to eradicate it. The combination of the use of Bti with SIT are most effective and selective tools against Ae. albopictus , one of the most dangerous mosquito vector species.

The recent global outbreaks of the ZIKA Virus (ZIKV) reported in 85 countries and territories caused severe complications such as microcephaly among neonates and Guillain-Barre Syndrome among the older population. Recently, an outbreak of ZIKV was reported from Thiruvananthapuram, the capital city of Kerala, India with 66 cases being reported so far. We conducted an outbreak investigation and the primary findings are described here. A cluster of ZIKV cases from the Kadakampalli / Anamugham administrative wards of the Thiruvananthapuram Municipal Corporation area was reported where Kerala Institute of Medical Sciences (KIMS) is located. Later many ZIKV cases were reported from other wards of the city. The density of known Aedes vectors was high in this region of the metropolitan city. Aedes albopictus , Aedes aegypti and Aedes vittatus collected from the focal area of the outbreak were found to be naturally infected with ZIKV. Male specimens of Ae. albopictus were naturally infected, indicating trans-ovarian transmission of the virus. This is first report of incrimination of Ae. albopictus and Ae. vittatus in ZIKV transmission from India. The virus was characterized and the partial sequences clustered with the Asian strain of ZIKV reported from India. The NS5 sequences of human and Ae. albopictus pools from Thiruvananthapuram were 100% similar indicating an ongoing active ZIKV transmission. The state health authorities were sensitized and appropriate containment and vector control measures have been initiated to contain the outbreak. This report underscores the importance of continued human and vector surveillance as well as genomic sequencing to understand the virus evolution and implications on public health.

SARS-CoV-2 (CoV2) is the viral agent responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Vaccines are being deployed all over the world with good efficacy, but there is no approved antiviral treatment to date. This is particularly needed since the emergence of variants and the potential immune escape may prolong pandemic spreading of the infection for much longer than anticipated. Here, we developed a series of small molecules and identified RG10 as a potent antiviral compound against SARS-CoV-2 in cell lines and human airway epithelia (HAE). RG10 localizes to endoplasmic reticulum (ER) membranes, perturbing ER morphology and inducing ER stress. Yet, RG10 does not associate with SARS-CoV-2 replication sites although preventing virus replication. To further investigate the antiviral properties of our compound, we developed fluorescent SARS-CoV-2 viral particles allowing us to track virus arrival to ER membranes. Live cell imaging of replication-competent virus infection revealed that RG10 stalls the intracellular virus-ER dynamics. Finally, we synthesized RG10b, a stable version of RG10, that showed increased potency in vitro and in HAE with a pharmacokinetic half-life greater than 2 h. Together, our work reports on a novel fluorescent virus model and innovative antiviral strategy consisting of the perturbation of ER/virus dynamics, highlighting the promising antiviral properties of RG10 and RG10b.

ABSTRACT Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity , has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility.

Mosquito-borne viruses including dengue, Zika and Chikungunya viruses as well as parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti (New Orleans strain) were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. Of the 400 compounds screened, we identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that significantly reduced Ae. aegypti larval motility compared to DMSO controls. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC50s of 25.5 ± 5.0 μM and 22.3 ± 5.4 μM respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to Ae. aegypti adults at 100 μM and 10 μM and completely blocked egg laying when fed at 100 μM. Camptothecin and its derivatives, inhibitors of topoisomerase I, have known activity against several agricultural pests and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries. Both humans and mosquitoes express the highly conserved topoisomerase I target, however, the design of derivatives with differing pharmacokinetic properties may offer a promising route towards the development of insect-specificity of this chemistry.

Acute febrile illnesses are still a major cause of mortality and morbidity globally, particularly in low to middle income countries. The aim of this study was to determine any possible metabolic commonalities of patients infected with disparate pathogens that cause fever. Three liquid chromatography-mass spectrometry (LC-MS) datasets investigating the metabolic effects of malaria, leishmaniasis and Zika virus infection were used. The retention time (RT) drift between the datasets was determined using landmarks obtained from the standard reference mixtures generally used in the quality control of the LC-MS experiments. We used fitted Gaussian Process models (GPs) to perform a high level correction of the RT drift between the experiments, followed by standard peakset alignment between the samples with corrected RTs of the three LC-MS datasets. Statistical analysis, annotation and pathway analysis of the integrated peaksets were subsequently performed. Metabolic dysregulation patterns common across the datasets were identified, with kynurenine pathway being the most affected pathway between all three fever-associated datasets.

ABSTRACT Zika virus (ZIKV) is a positive-sense RNA flavivirus and can cause serious neurological disorders including microcephaly in infected fetus. As a mosquito-borne arbovirus, ZIKV enters bloodstream and is transmitted into the fetus through the placenta in pregnant women. Monocytes are considered one of the earliest blood cell types to be infected by ZIKV. As a first line defence, monocytes are crucial components in innate immunity and host responses and may impact viral pathogenesis in humans. Previous studies have shown that ZIKV infection can activate inflammasomes and induce proinflammatory cytokines in monocytes. In this report, we showed that ZIKV carried out a productive infection, which lead to cell death in human and murine monocytic cells. In addition to the presence of cleaved caspase-3, indicating that apoptosis was involved, we identified the cleaved caspase-1 and gasdemin D (GSDMD) as well as increased secretion of IL-1β and IL-18, suggesting that the inflammasome was activated that may lead to pyroptosis in infected monocytes. The pyroptosis was NLRP3-dependent and could be suppressed in the monocytes treated with shRNA to target and knockdown caspase-1, or an inhibited for caspase-1, indicating that the pyroptosis was triggered via a canonical approach. Our findings in this study demonstrate a concomitant occurrence of apoptosis and pyroptosis in ZIKV-infected monocytes, with multiple mechanisms involved in the cell death, which may have potentially significant impacts on viral pathogenesis in humans.

ABSTRACT Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. Approximately 14% of the 817 identified proteins coincide with interactomes obtained in vertebrate cells, including ontology groups of the oligosaccharide transferase complex, the chaperonin containing TCP-1, and nuclear import and export, vesicle localization and ribosomal proteins. Notably, other protein pathways such as epigenetic regulation and RNA silencing, not previously reported in vertebrate cells, were also found in the NS1 interactome in mosquito cells. Due to the novel interaction observed for NS1 and DIDO1 (Death Inducer-Obliterator 1), we further explored the role of DIDO1 in viral replication. Interactions between NS1 and DIDO1were corroborated in infected C6/36 and Aag2 cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression in C6/36 and Aag2 cells results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected C6/36 silenced for DIDO1, revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response and necessary for flavivirus replication. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication and add to the understanding of NS1 as a multifunctional protein. ABSTRACT IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as epigenetic regulation and RNA silencing, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in vertebrate and mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1, revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

The 2015-2017 Zika virus (ZIKV) outbreak in Brazil and subsequent international epidemic revealed the strong association between ZIKV infection and congenital malformations. Vertical transmission leads with high frequency to congenital ZIKV syndrome (CZS). CZS is defined by neurodevelopmental and neurodegenerative defects, of which microcephaly is of the greatest concern. The scale and global expansion of the epidemic, the new ZIKV outbreaks (Kerala state, India, 2021), and the potential burden of future ones pose a serious ongoing risk. However, the mechanisms of congenital neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC and cortical neurons. The rapid proliferation of NPC requires efficient DNA damage responses to ensure the genome stability during neurodevelopment. Like congenital ZIKV infection, mutations in the polynucleotide 5’-kinase 3’-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, produce recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there are any links between ZIKV and PNKP. Here we show that the PNKP phosphatase inhibitor A12B4C3 inhibited ZIKV replication in NPC and non-neural cells. PNKP relocalized from the nucleus to the cytoplasm in infected cells, co-localizing with the marker of ZIKV replication factories (RF) NS1, resulting in functional nuclear PNKP depletion. Consequently, infected NPC accumulated DNA damage. However, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV infection induced accumulation and activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, suggesting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. The unscheduled mitotic entry in the presence of DNA damage results in mitotic catastrophe (MC). In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors playing critical roles in ZIKV replication.

ABSTRACT The causative agent of COVID-19 pandemic, the SARS-CoV-2 coronavirus, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 100 modified bases that are essential for proper function. Among internal modified bases, the N 6 -methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced so far are in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because incorporation of dNTPs hides RNA base modifications. Here, in this perspective paper, we present an initial exploration of the Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3’-untranslated region. We identified 15 m6A methylated positions, of which, 6 are in ORF N. Also, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants we show that variants Beta and Eta have a fourth position C>U change in DRACH at 28,884b that could affect methylation. The Nanopore technology offers a unique opportunity for the study of viral epitranscriptomics. This technique is PCR-free and is not sequencing-by-synthesis, therefore, no PCR bias and synthesis errors are introduced. The modified bases are preserved and assessed directly with no need for chemical treatments or antibodies. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

ABSTRACT The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. In particular, previous work evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both of these antiviral pathways. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti mosquitoes. This effect included a quantitatively greater reduction in salivary gland ZIKV levels relative to single pathway activation, indicating the potential for field delivery of these small molecules to substantially reduce virus transmission.

This paper explores the impact of the Covid-19 crisis at the level of individual information behavior, based on an online survey among 308 participants from the German-speaking countries Austria, Germany and Switzerland in April and May 2020. The results show first that the Covid-19 crisis has led to an increased demand for reliable information. This goes alongside a significant increased use of public broadcasting, newspapers and information provided by public organizations. Second, the majority (84%) of the participants reported being satisfied with the information supply during the Covid-19 crisis. Participants who were less satisfied with the information supply used reliable sources significantly less frequently, specifically public television, national newspapers and information provided by public organizations. Third, the amount of Covid-19-related information led some participants to a feeling of information overload, which resulted in a reduction of information seeking and media use.

Background: Real-time surveillance of search behavior on the Internet has achieved accessibility in measuring disease activity. Here, we systematically assessed the associations between Internet search trends of GI symptoms and daily newly confirmed COVID-19 cases at the global and country levels. Methods: Relative search volumes (RSVs) of the terms of GI symptoms were derived from Internet search engines. Time-series analyses with autoregressive integrated moving average models were conducted to fit and forecast the RSVs trends of each GI symptom before and after the COVID-19 outbreak. Generalized additive models were used to quantify the effects of RSVs of GI symptoms on the incidence of COVID-19. In addition, dose-response analyses were applied to estimate the shape of the associations. Findings: The RSVs of GI symptoms could be characterized by seasonal variation and high correlation with symptoms of fever and cough at worldwide and country levels; especially, “diarrhea” and “loss of taste” were abnormally increased during the outbreak period of COVID-19 with elevated point changes of 1.31 and 8 times, respectively. In addition, these symptom terms could effectively predict the COVID-19 outbreak in advance underlying lag correlation at 12 and 5 days, respectively, and with mutual independence as well. The dose-response curves showed a consistent increase in daily COVID-19 risk with increasing search volumes of “diarrhea” and “loss of taste”. Interpretation: Our comprehensive research was the first and largest infodemiological study that revealed the advanced prediction of the COVID-19 outbreak via GI symptom indicators. Funding: None to declare. Declaration of Interest: We declare no competing interests.

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of 7 wastewater samples. RT□LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best surrogate marker for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be used as a surrogate marker for detection of SARS-CoV-2. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except the one which subjected to RT-LAMP for targeting N region after RNA extraction (yellowish/orange color). This study shows for the first time that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips.

Dengue is the most prevalent arthropod-borne disease globally and affects approximately 2.5 billion people living in over 100 countries. The increasing geographic expansion of Aedes aegypti mosquitoes which transmit the virus has made dengue fever a global health concern. There are currently no approved antivirals available to treat dengue, and the only approved vaccine used in some countries is limited to seropositive patients. Treatment of dengue therefore remains largely supportive to date; hence research efforts are being intensified for the development of antivirals against dengue. The NS3 and NS5 nonstructural proteins have been the major targets for dengue antiviral development due to their indispensable enzymatic and biological functions in the viral replication process. NS5 is the largest and most conserved nonstructural protein encoded by flaviviruses including dengue. Its multifunctionality makes it an attractive target for antiviral development against dengue, but research efforts are hindered due to its limited structural characterization compared to the NS5 of other flaviviruses like the Zika virus. Increase in structural insights into the dengue NS5 protein will accelerate drug discovery efforts focused on NS5 as an antiviral target. In this review, we will give an overview of the current state of therapeutic development against dengue.

The emergence of Zika virus (ZIKV) infection, which is an unexpectedly associated with congenital defects, has prompted the development of safe and effective vaccines. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has emerged as a versatile and highly effective platform for delivering target proteins for vaccines. In this article, we developed a bacterium-like particle vaccine ZI-Δ-PA-GEM based on the GEM-PA system. The fusion protein ZI-Δ-PA, which contains the prM-E-Δ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3, was expressed. The fusion protein was successfully displayed on the GEM surface, forming ZI-Δ-PA-GEM. Moreover, when BALB/c mice were immunized intramuscularly with ZI-Δ-PA-GEM combined with 201 VG and poly(I:C) adjuvants, durable ZIKV-specific IgG and protective neutralizing antibody responses were induced. Potent B cell/DC activation was also be stimulated early after immunization. Remarkably, splenocyte proliferation, the secretion of multiple cytokines, T/B cell activation and central memory T cell responses were elicited. These data indicate that ZI-Δ-PA-GEM is a promising bacterium-like particle vaccine candidate for ZIKV. Author summary Because Zika virus (ZIKV) infection is considered as an example of “disease X”, the development of a safe and effective ZIKV vaccine is essential. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has been used in many vaccine studies due to its advantages. In this study, prM-E-△ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3 was fusion expressed, termed ZI-△-PA. Then the fusion protein ZI-△-PA could be displayed on the surface of GEM, forming ZI-△-PA-GEM. The author evaluated the immunogenicity of ZI-△-PA-GEM with the 201 VG and poly(I:C) adjuvants. The study demonstrates that ZI-△-PA-GEM induced mice to produce neutralizing antibody and specific cellular immune responses. The author believe that the bacterium-like particle vaccine ZI-△-PA-GEM has the potential to be used as the ZIKV vaccine.

Aedes aegypti mosquito has spread throughout the tropical and subtropical world and is currently the primary species responsible for transmitting dengue, urban yellow fever, Chikungunya, and Zika virus. This study aimed to investigate the inter- and intrapopulational genetic variability of Aedes aegypti through mitochondrial DNA, COI, ND4, and ND5 molecular markers in four municipalities in Mato Grosso. We used the Geneious software to build dendrograms for differentiating populations from each municipality. The interpopulational genetic distance obtained from sequence analysis showed a difference within populations through groups' formation in the ordering. Besides, we identified a difference in the interindividual genetic distance values, notably for the ND5 gene from the populations captured in the four municipalities. We recorded the smallest interindividual genetic distance within populations for populations from Chapada dos Guimarães. Extrinsic factors, including breeding habitat removal, can contribute to decreasing variability. Consequently, the dendrogram showed some similarities. Ovitrap monitoring, vector elimination, and genetic flow investigation stimulate actions to prevent transmitted diseases and support essential effective measures to control and fight Ae. aegypti .