Abstract It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare, spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback, and then barcoded for tracking via sequencing (i.e., Ran dom- De letion L ibrary seq uencing, RanDeL-seq). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis -acting elements (i.e., LTR, Ψ, and RRE) and surprisingly indicated that HIV-1’s central DNA flap (i.e., central polypurine tract, cPPT to central termination sequence, CTS) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis -acting regions are larger than the UTR termini, encompassing a large fraction of the non-structural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections. Importance Recent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)—a subset of viral deletion mutant that conditionally replicate. Identifying and engineering DIPs requires that viral cis - and trans -acting elements be accurately mapped. Here we introduce a high-throughput method (Random Deletion Library sequencing, RanDeL-seq) to comprehensively map cis- and trans -acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central poly-purine tract (cPPT) and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.

Exoribonuclease-resistant RNAs (xrRNAs) from viruses prevent digestion by host exoribonucleases, creating sub-genomic viral RNAs that can enhance infection and pathogenicity. Novel knotted structures in xrRNAs are proposed to act as mechanical road-blocks to RNases. Studying an xrRNA from Zika virus with optical tweezers, we found that it was the most mechanically stable RNA structure yet observed. The knot folded by threading the 5′ end into the cleft of a Mg 2+ -coordinated three-helix junction before pseudoknot interactions closed a ring around it. Both the threading and pseudoknot were required to generate the extremely force-resistant knot, whose formation correlated directly with RNase resistance both in the wild-type xrRNA and a low-resistance mutant. This work clarifies the folding and mechanism of action of an important new class of RNA.

Summary Paragraph Mosquito-borne viruses have recently spread globally, with major impacts on human health. Zika virus (ZIKV) emerged from obscurity in 2013 to spread from Asia to the South Pacific and the Americas, where millions of people were infected. For the first time, severe clinical manifestations, including Guillain Barré syndrome and congenital defects to the fetus of pregnant women, were detected. Phylogenetic studies have shown that ZIKV evolved in Africa and later spread to Asia, and that the Asian lineage is responsible for the recent epidemics. However, the reasons for the sudden emergence of ZIKV remain incompletely understood. Accumulating evidence on other arboviruses like chikungunya and West Nile suggest the likelihood that viral mutations could be determinants of change in ZIKV transmission efficiency responsible for efficient spread. Using evolutionary analyses, we determined that four mutations, which occurred just before ZIKV introduction to the Americas, represent direct reversions of previous mutations that accompanied spread many decades ago from ZIKV’s native Africa to Asia and early circulation there. Experimental infections of mosquitoes, human cells, and mice with ZIKV strains with and without these mutations demonstrated that the original mutations reduced fitness for urban transmission, while the reversions restored fitness, increasing epidemic risk. Our findings help to explain the recent ZIKV emergence and underscore the major yet unpredictable impacts of founder effects and genetic drift on viral emergence. Because they involve random mutations that become fixed in a population when it undergoes bottlenecks during geographic spread and inefficient transmission, founder effects and other forms of drift can limit the ability of mosquito-borne viruses like ZIKV to emerge. Furthermore, their stochastic nature limits our ability to predict epidemics.

In the aftermath of the 2015 pandemic of Zika virus, concerns over links between climate change and emerging arboviruses have become more pressing. Given the potential that much of the world might remain at risk from the virus, we use a model of thermal bounds on Zika virus (ZIKV) transmission to project climate change impacts on transmission suitability risk by mid-century (a generation into the future). In the worst-case scenario, over 1.3 billion new people could face suitable transmission temperatures for ZIKV by 2050. Given these suitability risk projections, we suggest an increased priority on research establishing the immune history of vulnerable populations, modeling when and where the next ZIKV outbreak might occur, evaluating the efficacy of conventional and novel intervention measures, and increasing surveillance efforts to prevent further expansion of ZIKV.

Abstract Background: Although it is known that Zika virus (ZIKV) infection during pregnancy may lead to microcephaly in the fetus, the prognostic factors associated with this tragic disorder remain unclear. We conducted a systematic review and meta-analysis to assess the prognostic factors associated with the incidence of microcephaly in congenital ZIKV infection. Methods: We conducted a comprehensive search in Ovid MEDLINE, Ovid MEDLINE (R) Epub ahead of print, Embase, Embase Classic, Web of Science, CINAHL, Cochrane CENTRAL, LILACS, and various thesis databases to identify human studies reporting microcephaly associated with congenital ZIKV infection. We requested primary data from the authors of the included studies to calculate summary estimates and conduct the meta-analysis of the most prevalent factors.Results: We screened 4106 titles and abstracts, and identified 12 studies for inclusion in the systematic review. The assessment of ZIKV infection and the definition of microcephaly varied among studies. A total of 6154 newborns/fetuses were enrolled; of those, 1120 (18.20%) had a diagnostic of ZIKV infection, of which 509 (45.45%) were diagnosed with microcephaly. Nine studies addressed the link between congenital ZIKV infection and neurological findings in newborns/fetuses. Half of the studies provided primary data. Three out of 11 factors of interest seem to be prognostic factors of microcephaly: infant’s sex – males compared to females: Relative Risk (RR) 1.30, 95% Confidence Interval (95% CI) 1.14 to 1.49; the stage of pregnancy when infection occurred – infection in the first trimester of pregnancy compared to infection at other stages of pregnancy: RR 1.41, 95% CI 1.09 to 1.82; and asymptomatic infection compared to symptomatic infection during pregnancy: RR 0.68; 95% CI 0.60 to 0.77.Conclusion: Our findings support the female-biased resistance hypothesis and reinforce the risk associated with the stage of pregnancy when ZIKV infection occurs. Continued surveillance of ZIKV infection during pregnancy is needed to identify additional factors that could contribute to developing microcephaly in affected fetuses.

ABSTRACT Viruses have developed innovative strategies to exploit the cellular machinery and overcome the host antiviral defenses, often using specifically structured RNA elements. Examples are found in flaviviruses; during flaviviral infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) accumulate in the cell. These sfRNAs are formed when a host cell 5’ to 3’ exoribonuclease degrades the viral genomic RNA but is blocked by an exoribonuclease resistant RNA structure (xrRNA) located in the viral genome’s 3’untranslated region (UTR). Although known to exist in several Flaviviridae genera the full distribution and diversity of xRNAs in this virus family was unknown. Using the recent high-resolution structure of an xrRNA from the divergent flavivirus Tamana bat virus (TABV) as a reference, we used bioinformatic searches to identify xrRNA in the Pegivirus, Pestivirus, and Hepacivirus genera. We biochemically and structurally characterized several examples, determining that they are genuine xrRNAs with a conserved fold. These new xrRNAs look superficially similar to the previously described xrRNAs but possess structural differences making them distinct from previous classes of xrRNAs. Our findings thus require adjustments of previous xrRNA classification schemes and expand on the previously known distribution of the xrRNA in Flaviviridae , indicating their widespread distribution and illustrating their importance. IMPORTANCE The Flaviviridae comprise one of the largest families of positive sense single stranded (+ssRNA) and it is divided into the Flavivirus , Pestivirus , Pegivirus , and Hepacivirus genera. The genus Flavivirus contains many medically relevant viruses such as Zika Virus, Dengue Virus, and Powassan Virus. In these, a part of the virus’s RNA twists up into a very special three-dimensional shape called an xrRNA that blocks the ability of the cell to “chew up” the viral RNA. Hence, part of the virus’ RNA remains intact, and this protected part is important for viral infection. This was known to occur in Flaviviruses but whether it existed in the other members of the family was not known. In this study, we not only identified a new subclass of xrRNA found in Flavivirus but also in the remaining three genera. The fact that this process of viral RNA maturation exists throughout the entire Flaviviridae family makes it clear that this is an important but underappreciated part of the infection strategy of these diverse human pathogens.

III. Abstract The Zika virus (ZIKV) of the Flaviviridae family is an emerging virus that caused, between 2016 and 2018, serious public health problems in Latin America, affecting neonates with greater severity. The clinical spectrum includes Guillain-Barré syndrome, microcephaly and others neurodegenerative diseases. There is no antiviral treatment or vaccine against this virus, for that reason the antiviral properties of various plants are being studied. Lippia alba , locally known as “Prontoalivio”, is an aromatic shrub of the Verbenaceae family with a wide geographical distribution (especially in South and Central America) and is used in traditional medicine against fever, skin diseases and as a pain reliever. In this study, the antiviral activity of the essential oil of Lippia alba against ZIKV was evaluated in the Vero 76 cell line. Lippia alba was collected in the department of Amazonas, in the rainforest of Peru, and identified in the Museo de Historia Natural of the Universidad Nacional Mayor de San Marcos. The essential oil sample was obtained by steam hydrodistillation. The essential oil showed cytotoxicity to a concentration greater than or equal to 167 μg/mL in the Vero 76 cell line. The antiviral activity of essential oil against ZIKV (previously identified by real-time PCR and propagated in the C6/36 cell line) was evaluated using the plaque reduction test (PRP). The essential oil showed antiviral activity in concentrations from 8.02 μg/mL to 20.88 μg/mL, which represents a range between 59.44% to 85.56% of plaque reduction and may be considered as a candidate for antiviral studies against ZIKV. IV. Author Summary Environmental temperature fluctuations, human activities and vector characteristics increase ZIKV cases worldwide. This neglected disease has been silently affecting people of all ages, generating greater impact on neonates by causing microcephaly and other neurodegenerative diseases. ZIKV vaccines are in phase II trials and there is no antiviral treatment. Nowadays, the study of antiviral plants is gaining strength in the scientific community because they are known to contain chemical compounds that could be drugs candidates. In Peru, there are no antiviral treatments studies reported against Zika virus, this first report is important because it creates a new line of research for future studies of antiviral plant extracts against neglected viral-diseases. Finally, the essential oil of Lippia alba showed antiviral activity against ZIKV in Vero 76 cells, moreover, we intend to carefully isolate and study the chemical compounds as drug candidates in animals trials and possible humans trials. Disclaimer The views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. Copyright statement Juan Sulca is an employee of the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that ‘Copyright protection under this title is not available for any work of the United States Government’. Title 17 U.S.C. § 101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties.

Background . Outbreaks of mosquito-borne arboviral diseases including dengue virus (DENV), Zika virus (ZIKV), yellow fever virus (YFV) and chikungunya virus (CHIKV) have recently occurred in the Caribbean. The geographical range of the principle vectors responsible for transmission, Aedes (Ae.) aegypti and Ae . albopictus is increasing and greater mosquito surveillance is needed in the Caribbean given international tourism is so prominent. The island of Saint Lucia has seen outbreaks of DENV and CHIKV in the past five years but vector surveillance has been limited with the last studies dating back to the late 1970s. Natural disasters have changed the landscape of Saint Lucia and the island has gone through significant urbanisation. Methods . In this study, we conducted an entomological survey of Ae. aegypti and Ae. albopictus distribution across the island and analysed environmental parameters associated with the presence of these species in addition to screening for medically important arboviruses and other flaviviruses. Results. Although we collected Ae. aegypti across a range of sites across the island, no Ae. albopictus were collected despite traps being placed in diverse ecological settings. The number of Ae. aegypti collected was significantly associated with higher elevation, and semi-urban settings yielded female mosquito counts per trap-day that were five-fold lower than urban settings. Screening for arboviruses revealed a high prevalence of a novel insect-specific flavivirus closely related to cell fusing agent virus (CFAV). Conclusions. Outbreaks of arboviruses transmitted by Ae. aegypti and Ae. albopictus have a history of occurring in small tropical islands and Saint Lucia is particularly vulnerable given the limited resources available to undertake vector control and manage outbreaks. Surveillance strategies can identify risk areas for predicting future outbreaks and further research is needed to determine the diversity of current mosquito species and this should be extended to the neighbouring smaller Caribbean islands.

Abstract Background Since the huge epidemic of Zika virus (ZIKV) in Brazil in 2015, questions were raised to understand which mosquito species could transmit the virus. Aedes aegypti has been described as the main vector. However, other Aedes species (e.g. Ae. albopictus and Ae. japonicus ) proven to be competent for other Flaviviruses (e.g.West Nile, dengue and yellow fever), have been described as potential vector for ZIKV under laboratory conditions. To name one, the Asian bush mosquito Ae. japonicus which is widely distributed with high abundances in Central-Western Europe. In the present study, infection, dissemination and transmission rates of ZIKV (Dak84 strain) in two populations of Ae. japonicus from Switzerland (Zürich) and France (Steinbach, Haut-Rhin) were investigated under constant (27 °C) and fluctuating (14-27 °C, mean 23 °C) temperature regimes. Results The two populations were each able to transmit ZIKV under both temperature regimes. Infectious virus particles were detected in the saliva of females from both populations, regardless of the incubation temperature regime, from 7 days post exposure to infectious rabbit blood. The highest amount of Plaque Forming Unit (PFU) (400/ml) were recorded 14 days post oral infection in the Swiss population incubated at constant temperature. No difference in term of infection, dissemination and transmission rate were found between mosquito populations.Temperature had no effect on infection rate but the fluctuating temperature regime resulted in higher dissemination rates comparing to constant temperature, regardless of the population. Finally, transmission efficiency ranged between 7-23% and 7-10% for the constant temperature and 0-10% and 3-27% under fluctuating temperatures for the Swiss and the French populations, respectively. Conclusions This is the first work confirming vector competence for ZIKV of Ae. japonicus originating from Switzerland and France at realistic summer temperatures under laboratory conditions. Considering the continuous spread of this species in the northern part of Europe and its adaptation at cooler temperatures, preventative control measures should be adopted to prevent possible ZIKV epidemics.

Abstract Background In 2016 and 2017, Zika virus (ZIKV) infection outbreaks occurred in two communities in southern Thailand. This re-immerging infection can widely spread by mosquito bites and cause serious complications in a central nervous system, especially in babies. Thus they should be protected. This study aims to (1) To determine the prevalence of neutralizing ZIKV antibodies in the post-outbreak areas among the general population and pregnancy women residing at various distances from the houses of the nearest index patients; (2) To examine the cross-neutralizing capacity of antibodies against ZIKV on other flaviviruses commonly found in the study areas; (3) To identify factors associated with the presence of neutralizing ZIKV antibodies. Methods The two post-outbreak communities were visited at 18 months after the outbreaks. We enrolled (1) 18 confirmed ZIKV infected (index) cases, (2) sample of 554 neighbors in the outbreak areas who lived at various distances from the index patients’ houses, (3) 190 residents of non-outbreak areas, and (4) all pregnant women regardless of gestational age residing in the study areas (n = 805). All serum specimens underwent the plaque reduction neutralization test (PRNT). Ten randomly selected ZIKV seropositive and ten randomly selected seronegative specimens were tested for dengue virus serotypes 1-4 (DENV1-4) and Japanese encephalitis virus (JEV) antibodies using PRNT. Serum titer above 1:10 was considered positive. Multiple logistic regression was used to assess factors associated with seropositivity. Results Out of all 18 index cases, 17 remained seropositive. The seroprevalence (95% CI) in the two outbreak areas were 66.5% (59.1-73.9%) and 45.6% (38.8-52.5%) in general population, and 41.0% (36.1-46.0%) and 23.5% (19.4-21.9%) in pregnant women. Multivariate analysis showed that seropositivity was independent of the distance gradient from the index’s houses. However, being elderly, poorly educated, and living far from a plantation was associated with seropositivity. DENV1-4 and JEV neutralizing antibodies were present in nearly all ZIKV-positive and negative subsamples. Conclusion Protective herd immunity for ZIKV infection is inadequate, especially among pregnant women in the two post-outbreak areas in southern Thailand.

Abstract The SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The data available about COVID-19 during pregnancy have demonstrated placental infection; however, the intrauterine transmission of SARS-CoV-2 is still debated. Intriguingly, while canonical SARS-CoV-2 cell entry mediators are expressed at low levels in placental cells, the receptors for viruses that cause congenital infections such as the cytomegalovirus and Zika virus are highly expressed in these cells. Here we analyzed the transcriptional profile (microarray and single-cell RNA-Seq) of proteins potentially interacting with coronaviruses to identify non-canonical mediators of SARS-CoV-2 infection and replication in the placenta. We show that, despite low levels of the canonical cell entry mediators ACE2 and TMPRSS2 , villous trophoblast cells co-express high levels of the potential non-canonical cell-entry mediators DPP4 and CTSL . We also found changes in the expression of DAAM1 and PAICS genes during pregnancy, which are translated into proteins also predicted to interact with coronaviruses proteins. These results provide new insight into the interaction between SARS-CoV-2 and host proteins that may act as non-canonical routes for SARS-CoV-2 infection and replication in the placenta cells.

Abstract The recent spread of Zika virus (ZIKV) and its association with congenital defects and neurological disorders has created an urgent need to understand the pathogenesis of ZIKV and identify therapeutic strategies that will prevent or eliminate them. The neurodevelopmental defects associated with ZikV infections early in pregnancy are well documented, however the potential defects associated with infections in late pregnancy and perinatal period are less well characterized. Further, the long-term sequelae of these infections are not fully understood. Immunocompetent C57BL/6 mice infected at one day old (P1), which neurodevelopmentally model late pregnancy in humans, develop a transient neurological syndrome including unsteady gait, kinetic tremors, severe ataxia and seizures 10-15 days post-infection (dpi) but symptoms subside after a week, and most animals survive. Despite apparent recovery, MRI of convalescent mice shows reduced cerebellar volume that correlates with altered coordination and motor function as well as hyperactivity and impulsivity. Persistent mRNA levels of pro-inflammatory genes including Cd80, Il-1α , and Ifn-γ together with Cd3, Cd8 and perforin ( PrfA) , suggested the persistence of a low-grade inflammatory process. Further, the brain parenchyma of convalescent mice harbor multiple small foci with viral antigen, active apoptotic processes in neurons, and cellular infiltrates, surrounded by activated astrocytes and microglia as late as 1-year post-infection. Detection of negative-sense strand viral RNA and replication of virus derived from these convalescent mice by blinded passage in Vero cells confirmed that low levels of live ZikV persist in CNS of convalescent mice life-long. Our studies establish that Zika can establish reservoirs in CNS and suggest that anti-viral treatment that clears virus from the CNS as well as long-term neurological and behavioral monitoring may be needed for patients known to be exposed to ZikV at an early age. Author’s summary The congenital brain malformations associated with ZikV infections early in pregnancy are well documented, however whether apparently asymptomatic perinatal exposure could lead to long term sequelae is not fully understood. Using a non-lethal neonatal mouse model, we examine host-pathogen interactions, anatomical changes and behavioral patterns by following survivors of the acute infection for over 1 year. We discover that infectious Zika virus has the potential to remain in the CNS for life, lodged within small foci surrounded by gliosis and infiltrating immune cells that may act to limit the viral spread, but also interfere with healing and contribute to life-long neuropathic and behavioral sequelae. These results suggest that anti-viral treatment and long-term neurological and behavioral monitoring may be indicated for patients known to have been exposed to Zika virus, regardless of neurodevelopmental disease severity.

Abstract Mosquito-transmitted flaviviruses, such as Dengue virus (DENV) or Zika virus (ZIKV), are responsible for significant economic damage and human misery. In infected cells, flaviviruses first assemble into an immature form within the endoplasmatic reticulum (ER), and then undergo further processing by furin protease in the trans-Golgi. Despite substantial efforts, previous cryogenic electron microscopy (cryo-EM) studies of immature flaviviruses were restricted to low to medium resolutions, limiting our understanding of maturation. To better grasp the process of maturation, we have carried out cryo-EM reconstructions of immature Spondweni virus (SPOV), an emerging human flavivirus belonging to the same serogroup as ZIKV (~75% amino acid identity). By combining localized reconstruction and focused refinement, we were able to improve the resolution to 3.8 Å, yielding unprecedented insight into the immature form. The structure elucidates how, at neutral pH, polar interactions conceal the furin recognition site within trimeric envelope (E) protein spikes. Furthermore, we identify how a strictly conserved pH sensor anchors the precursor membrane (prM) protein to immature E. We reconstructed mature forms of SPONV and DENV to 2.6Å and 3.1Å, respectively. Comparison with immature virus shows a conserved binding pocket for a lipid headgroup, which forms as a consequence of the rearrangement of amphipathic stem-helices of E. We propose a structural role for the pocket and suggest it stabilizes mature E. Taken together, our data suggest a compelling rationale for low-pH triggered conformational rearrangement in the Golgi, which occurs during flavivirus maturation.

Abstract West Nile virus (WNV) is a prominent mosquito-borne flavivirus that causes febrile illness in humans. To infect host cells, WNV virions first bind to plasma membrane receptors, then initiate membrane fusion following endocytosis. The viral transmembrane E protein, triggered by endosomal pH, catalyzes fusion while undergoing a dimer-to-trimer transition. Previously, single-particle WNV fusion data was interrogated with a stochastic cellular automaton simulation, which modeled the E proteins during the fusion process. The results supported a linear fusion mechanism, with E protein trimerization being rate-limiting. Here, we present corrections to the previous simulation, and apply them to the WNV fusion data. We observe that a linear mechanism is no longer sufficient to fit the data. Instead, an off-pathway state is necessary; these results are corroborated by chemical kinetics modeling. When compared with a similar Zika virus fusion model, this suggests that off-pathway fusion mechanisms may characterize flaviviruses more broadly.

Abstract Background There is substantial evidence of the profound consequences of Zika on women's Sexual and Reproductive Health. Health system resilience begins by measuring critical capacities ahead of a crisis such as Zika outbreak. Even though Zika as vector-borne disease is well documented, there is dearth of studies linking Zika with women's Sexual and Reproductive Health. The main objective of this study was to analyze the national response to the Zika epidemic and its relation to women’s sexual and reproductive health matters through key implementation mechanisms in order to promote resilience of the health system in five cities in Colombia. Methods This study used a qualitative design to enable an in-depth exploration of the national response to the Zika epidemic and sexual and reproductive health matters through key implementation mechanisms (based on facilitators and gaps) within the health system. The overall data set was comprised of 31 semi-structured individual interviews (23 women and 8 men), 25 interviews with key informants responsible for the implementation of the Zika Virus Response Plan; six interviews with pregnant women diagnosed with Zika; and five focus groups discussions with communities (n=122 participants) in five cities in Colombia: Barranquilla, Cucuta, Los Patios, San Andres and Soledad. Results The findings revealed the three major facilitators that promoted the implementation of actions to address the Zika epidemic: i) the role of health care providers; ii) the development of technical equipment capabilities; and iii) inter-institutional coordination. The study also identified implementation gaps: i) absence of a human rights and sexual and reproductive health approach; ii) focus on territorial actions centered on mosquito management; and iii) limited attitudes, behaviors and knowledge at the community level. Conclusion This study provided a comprehensive insight of critical facilitating processes and gaps in the implementation of the government response during the Zika epidemic in Colombia. This study reveals that the lack of understanding of the intersection between gender, the Zika epidemic and Sexual and Reproductive Health limited the adoption, development and implementation of a more comprehensive responses to address the impact on women’s sexual and reproductive health.

Abstract Background: Zika fever is a mosquito-borne infection induced by Zika virus (ZIKV),effective drug and vaccine against ZIKV infection is still unavailable. Cinnamomi ramulus (CR) is a traditional Chinese herb with a long history ,can stimulate secretion of sweat and relieve exterior syndrome. The aim of this research was to evaluate the effect of CR against ZIKV and uncover its mechanism of action by network pharmacology.Methods: Cell viability assay, qRT-PCR assay and western blot assays were perfermed to evaluate anti-ZIKV activity in vitro. Survival rate, body weight were observed and viremia was detected in AG6 mice. Drug- target-disease networks, GO enrichment, and KEGG pathway analysis was established to clarify the therapeutic mechanismsits. Results: In this study, we find that CR can alleviate CPE after Zika virus infection. In the premixed administration mode, CR showed superior activity to inhibit viral RNA replication and protein expression in cells. Orally administered CR effectively protected AG6 mice infected with lethal doses of ZIKV, conferring 50% or 20% survival rate at a dosage of 900mg/ml or 450mg/ml, reducing body weight loss, inhibiting viral RNA replication. Beta-sitosterol, DBP, α-Longipinen, (-)-alpha-cedrene, ()-alpha-Longipinene may the main active compound and PTGS2, GABRA1, PTGS1, PTGES and CCR5 may the main targets. 176 biological processes, 16 cell components, 27 molecular functions, and 37 pathways were significantly identified in GO enrichment and pathway analysis. Conclusions: These results reveal that CR treatment of Zika fever is mainly related to PTGS1 and PTGS2 regulated prostaglandin release, metabolism, and inflammation response.

Abstract Background Mosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases often relies on management of the vector. Traditional control methods such as source reduction and chemical insecticides, have proven not to be sufficient to prevent the proliferation and spread of mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males that would induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined. Methods We evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile (WNV) virus and Zika virus (ZIKV). Results CHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1, 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed predicting the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection. The corresponding equation uses a Ct value of 36 as a cut-off value for virus detection and a virus copy number of 10 8 for the positive mosquito body. Based on this formula, the detection limits of CHIK, USUV, ZIKV and WNV were 5.08 x 10 5 , 8.74 x 10 6 , 2.33 x 10 7 , and 5.24 x 10 5 , respectively. Conclusion Mosquito borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have an SIT component.

Abstract Background The Zika virus (ZIKV) outbreak that occurred in multiple countries was linked to increased risk of neurological disorders and congenital defects. However, host immunity and immune-mediated pathogenesis in ZIKV infection are not well understood. Interleukin-22 (IL-22) is a crucial cytokine for regulating host immunity in infectious diseases. Whether IL-22 plays a role in ZIKV infection is unknown. Methods The cellular source of IL-22 was identified in IFNAR −/− mice and WT neonatal mice during ZIKV infection. To determine the role of IL-22, we challenged 1-day-old wild-type (WT) and IL-22 −/− mice with ZIKV and monitored clinical manifestations. Glial cell activation in the brain was assessed by confocal imaging. ZIKV-specific CD8 + T cell responses in both the spleen and brain were analyzed by flow cytometry. In addition, we infected mouse primary astrocytes in vitro , and characterized the reactive astrocyte phenotype. Human glial cell line was also infected with ZIKV in the presence of IL-22, followed by the evaluation of cell proliferation, cytokine expression and viral loads. Results We found that γδ T cells were the main source of IL-22 during ZIKV infection in both the spleen and brain. WT mice began to develop weight loss, staggered steps, bilateral hind limb paralysis, weakness at 10 days post-infection (dpi), and ultimately succumbed to infection at 16–19 dpi. Surprisingly, IL-22 deficiency lessened weight loss, moderated the systemic inflammatory response, and greatly reduced the incidence of neurological disorders and mortality. ZIKV infection facilitated a neurotoxic polarization of A1-prone astrocytes in vitro . Additional analysis demonstrated that the absence of IL-22 resulted in reduced activation of microglia and astrocytes in the cortex. Although IL-22 displayed a marginal effect on glial cells in vitro , IL-22 −/− mice mounted more vigorous ZIKV-specific CD8 + T cell responses, which led to a more effective control of ZIKV in the brain. Conclusions Our data revealed a pathogenic role of IL-22 in ZIKV encephalitis.

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg 2+ and the key tertiary interactions. The absence of Mg 2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structure in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations on ZIKV xrRNA1 and another two RNAs with ring structures, the HCV IRES and THF riboswitch. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.

Abstract Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here we demonstrate that the NRF2 anti-oxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a potent cellular anti-viral program, which potently inhibits replication of SARS-CoV2 across cell lines. The anti-viral program extended to inhibit the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, induction of NRF2 by 4-OI and DMF limited host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2. One Sentence Summary: NRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate inhibited SARS-CoV2 replication and virus-induced inflammatory responses, as well as replication of other human pathogenic viruses.

Zika virus (ZIKV) is a flavivirus that originated in Africa but emerged in Latin America in 2015. In this region, other flaviviruses such as Dengue (DENV), West Nile, and Yellow Fever Virus (YFV) also circulate, allowing for possible antigenic cross-reactivity to impact viral infections and immune responses. Studies have found antibody mediated enhancement between DENV and ZIKV, but the impact of YFV antibodies on ZIKV infection has not been fully explored. ZIKV infections cause congenital syndromes, such as microcephaly, necessitating further research into ZIKV vertical transmission through the placental barrier. Recent advancements in biomedical engineering have generated co-culture methods that allow for in vitro recapitulation of the maternal: fetal interface. This study utilized a transwell assay, which is a co-culture model utilizing human placental syncytiotrophoblasts, fetal umbilical cells, and a differentiating embryoid body to replicate the maternal: fetal axis. To determine if cross reactive YFV vaccine antibodies impact the pathogenesis of ZIKV across the maternal fetal axis, maternal syncytiotrophoblasts were inoculated with ZIKV or ZIKV incubated with YFV vaccine anti-sera, and viral load was measured 72 hours post inoculation. The data show that the impact of YFV on ZIKV replication is cell line dependent. In differentiating embryoids, the presence of YFV antibodies enhanced ZIKV infection. Since viral pathogenesis, and the impact of antigenic cross-reactive antibodies, is cell line specific at the maternal-fetal axis, this suggests there may be discreet mechanisms that impact congenital ZIKV infections.

Summary The generation of in vitro model of human peripheral myelin development and associated disease from human pluripotent stem cells (hPSCs) has been a challenge so far. In addition, the underlying mechanism for ZIKA virus (ZIKV) infection incurred Guillain-Barré syndrome (GBS) remains unexplored due to the lack of a suitable model. Here, we report the de novo generation of a human peripheral myelination model with competent Schwann cells (SCs). Those human SCs generated from hPSCs via compound screening were capable of forming compact myelin both in vitro and in vivo . We found ZIKV infection caused GBS-like events in vitro including myelin sheath degeneration, as well as dysregulated transcriptional profile including the activated cell death pathways and cytokine production. These effects could be partially reversed by several pharmacological inhibitors. Our model therefore provides a new and robust tool for studying the pathogenic mechanisms and developing of therapeutic strategies for related neuropathies.

Abstract Viruses are the major aetiological agents of acute and chronic severe human diseases that place a tremendous burden on global public health and economy; however, for most viruses, effective prophylactics and therapeutics are lacking, in particular, broad-spectrum antiviral agents. Herein, we identified 2 secreted bacterial lipases from a Chromobacterium bacterium, named Chromobacterium antiviral effector-1 ( Cb AE-1) and Cb AE-2, with a broad-spectrum virucidal activity against dengue virus (DENV), Zika virus (ZIKV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus (HIV) and herpes simplex virus (HSV). The Cb AEs potently blocked viral infection in the extracellular milieu through their lipase activity. Mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. A mutation of Cb AE-1 in its lipase motif fully abrogated the virucidal ability. Furthermore, Cb AE-2 presented low toxicity in vivo and in vitro , highlighting its potential as a broad-spectrum antiviral drug.

Abstract COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within two days of exposure. Using COVID-19 convalescent serum, we identified that SARS-CoV-2 preferably targets soma of cortical neurons but not neural stem cells, the target cell type of ZIKA virus. Imaging cortical neurons of organoids reveal that SARS-CoV-2 exposure is associated with missorted Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Surprisingly, SARS-CoV-2 co-localizes specifically with Tau phosphorylated at Threonine-231 in the soma, indicative of early neurodegeneration-like effects. Our studies, therefore, provide initial insights into the impact of SARS-CoV-2 as a neurotropic virus and emphasize that brain organoids could model CNS pathologies of COVID-19. One sentence summary COVID-19 modeling in human brain organoids

ABSTRACT The pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected over 3.8 million people, including pregnant women. To date, no consistent evidence of vertical transmission for SARS-CoV-2 exists. This new coronavirus canonically utilizes the angiotensin-converting enzyme 2 (ACE2) receptor and the serine protease TMPRSS2 for cell entry. Herein, building upon our previous single cell study of the placenta (Pique-Regi, 2019), another study, and new single-cell/nuclei RNA-sequencing data, we investigated the expression of ACE2 and TMPRSS2 throughout pregnancy as well as in third-trimester chorioamniotic membranes. We report that co-transcription of ACE2 and TMPRSS2 is negligible, thus not a likely path of vertical transmission for SARS-CoV-2 at any stage of pregnancy. In contrast, receptors for Zika virus and cytomegalovirus which cause congenital infections are highly expressed by placental cell types. These data suggest that SARS-CoV-2 is unlikely to infect the human placenta through the canonical cell entry mediators; yet, other interacting proteins could still play a role in the viral infection.