Zika virus (ZIKV) and chikungunya virus (CHIKV) were recently introduced into the Americas resulting in significant disease burdens. Understanding their spatial and temporal dynamics at the subnational level is key to informing surveillance and preparedness for future epidemics. We analyzed anonymized line list data on approximately 105,000 Zika virus disease and 412,000 chikungunya fever suspected and laboratory-confirmed cases during the 2014-2017 epidemics. We first determined the week of invasion in each city. Out of 1,122, 288 cities met criteria for epidemic invasion by ZIKA and 338 cities by CHIKV. We estimated that the geographic origin of both epidemics was located in Barranquilla, north Colombia. Using gravity models, we assessed the spatial and temporal invasion dynamics of both viruses to analyze transmission between cities. Invasion risk was best captured when accounting for geographic distance and intermediate levels of density dependence. Although a few long-distance invasion events occurred at the beginning of the epidemics, an estimated distance power of 1.7 (95% CrI: 1.5-2.0) suggests that spatial spread was primarily driven by short-distance transmission. Cities with large populations were more likely to spread disease than cities with smaller populations. Similarities between the epidemics included having the same estimated geographic origin and having the same five parameters estimated in the best-fitting models. ZIKV spread considerably faster than CHIKV.

Abstract Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths derived from the viral 3’ untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of incomplete viral genome degradation of the 3’UTR by host 5’ to 3’ exoribonuclease (Xrn1). The halting of Xrn1 in the 3’UTR is due to two RNA pseudoknot structures in the 3’UTR termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we rescued a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3’ UTR structure. We found that virus which lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult IFNAR1 -/- mice, we found that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to WT ZIKV infection. Despite thee attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches.

Abstract Background: The implications of congenital Zika Virus (ZIKV) infections for pediatric neurodevelopment and behavior remain inadequately studied. The aim of this study is to investigate patterns of neurodevelopment and behavior in groups of children with differening severities of ZIKV-related microcephaly and children with prenatal ZIKV exposure in the absence of microcephaly. Methods: We conducted a cross-sectional study, nested in a cohort, of 274 children (aged 10-45 months) who were born during the peak and decline of the microcephaly epidemic in Northeast Brazil. Participants were evaluated between February 2017 and August 2019 at two tertiary care hospitals in Recife, Pernambuco, Brazil. We analyzed the children in four groups assigned based on clinical and laboratory criteria: Group 1 had severe microcephaly; Group 2 had moderate microcephaly; Group 3 had prenatal ZIKVexposure confirmed by maternal RT-PCR testing but no microcephaly; and Group 4 was a neurotypical control group. Groups were evaluated clinically for neurological abnormalities and compared using the Survey of Wellbeing of Young Children (SWYC), a neurodevelopment and behavior screening instrument validated for use in Brazil. Children with severe delays underwent further evaluation with an adapted version of the SWYC. Results: Based on the SWYC screening, we observed differences between the groups for developmental milestones but not behavior. Among the 114 children with severe microcephaly of whom 98.2% presented with neurological abnormalities, 99.1% were ‘at risk of development delay’ according to the SWYC instrument. Among the 20 children with moderate microcephaly of whom 60% presented with neurological abnormalities, 65% were ‘at risk of development delay’. For children without microcephaly, the percentages found to be ‘at risk of developmental delay’ were markedly lower and did not differ by prenatal ZIKV exposure status: Group 3 (N=94), 13.8%; Group 4 (N=46), 21.7%. Conclusions: Among children with prenatal ZIKV exposure, we found a gradient of risk of development delay according to head circumference. Children with severe microcephaly were at highest risk for delays, while normocephalic ZIKV-exposed children had similar risks to unexposed control children. We propose that ZIKV-exposed children should undergo first-line screening for neurodevelopment and behavior using the SWYC instrument. Early assessment and follow-up will enable at-risk children to be referred to a more comprehensive developmental evaluation and to multidisciplinary care management.

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 vectors for ZIKV under laboratory conditions. One of these, the Asian bush mosquito, Ae. japonicus , 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 units (PFU) (400/ml) were recorded 14 days post-oral infection in the Swiss population incubated at a constant temperature. No difference in terms 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 compared 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: To the best of our knowledge, this is the first study 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.

RNA viruses are responsible for many types of zoonotic diseases that post great challenges for public health system. Effective therapeutics against these viral infections remains limited. Here we deployed a computational framework for host-based drug repositioning to predict potential antiviral drug candidates from 2352 approved drugs and 1062 natural compounds embedded in Traditional Chinese Medicine herbs. By systematically interrogating public genetic screening data, we comprehensively catalogued human-specific host dependency genes that are indispensable for the successful viral infection corresponding to 10 families and 29 species of RNA viruses. In addition, we utilized these host dependency genes as potential drug targets, and interrogated extensive drug-target interactions through multiple ways such as database retrieval, literature mining and de novo prediction using artificial intelligence-based algorithms. Repurposed drugs or natural compounds were proposed for combating many viral pathogens such as coronaviruses (e.g., SARS-CoV-2), flaviviruses (e.g., Zika virus) and influenza viruses. This study helps to prioritize promising drug candidates for further therapeutic evaluation against these viral-related diseases.

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 primarily relies on the management of the vector. Traditional control methods are insufficient to control 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 with the aim to induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as the 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 virus (WNV) 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 and 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed the prediction of the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection. Conclusions: 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 a SIT component by reducing the time and the manpower needed to conduct this quality control process.

Study question: Does the fear of the coronavirus disease 2019 (COVID-19) pandemic reduce the desire of infertile couples to continue treatment? Summary answer: Most of the participants in this study wanted to continue treatment. What is known already: The effect of the prevalence of infectious diseases including the Zika virus on the attitude of infertile couples has been studied in very few studies. However, the effect of the outbreak of COVID-19 on the attitude of infertile couples has not been investigated. Study design, size, duration: We conducted a prospective longitudinal study on forty-six infertile couples (n=92) who referred to our infertility clinic from 4 March 2020 through 20 June 2020. Participants/materials, settings, methods: This study is based on potential infertile couples for treatment with assisted reproductive technology (ART) who referred to our infertility clinic, Sari, Iran (median age of 35.5 years). All individuals with primary infertility, as defined by the World Health Organization (WHO) and candidates for ART, were included in the study. People who did not agree to participate in the study were excluded. Subjects were surveyed using a researcher-made questionnaire. This questionnaire has four sections as follows: The first part included demographic information and clinical characteristics, the second part included medical records, the third part included questions related to assessing the level of awareness regarding coronavirus infection, and the fourth part included questions related to the attitude towards continuing infertility treatment. The validity of this questionnaire was assessed by three infertility specialists and was confirmed with Cronbachs alpha of 0.78. A P-value of less than 0.05 was considered statistically significant. Main results and the role of chance: There is no significant relationship between COVID-19 symptoms and the level of awareness (P-value <0.05). Thirty-two patients (33.33%) had decreased motivation to continue treatment during COVID-19 pandemic. Fear of transmission to the fetus (28.13%) had the highest frequency among the causes of decreased motivation to continue treatment (P-value = 0.011). Confidence in support of the treatment team (56.67%) was the most common reason for lack of motivation in people without decreased motivation (P-value <0.001). The majority of patients had a very high or high tendency (65.22%) to continue or start treatment during the COVID-19 pandemic (P-value <0.001). Most people had an average fear of getting the disease (39.13%) (value <0.001). Examining the relationship between the presence of COVID-19 symptoms and attitude level variables showed that there is only a significant relationship between the greater desire to have a child and the continuation or initiation of treatment with the presence of COVID-19 symptoms (P-value = 0.032). Limitations, reasons for caution: We were not able to fully assess patients' deep feelings and cultural beliefs, due to the use of questionnaires and the lack of interviews. Wider implications of the findings: Our results showed for the first time that patients' knowledge about COVID-19 and trust in the treatment staff played an effective role in selecting and continuing infertility treatment. To clarify this issue, studies with the larger statistical community in the form of interviews are needed. Study funding/completing interest(s): The study received financial support from the Mazandaran University of Medical Sciences (Grant number: 7903). None of the funders had any role in the study design, collection, analysis, or interpretation of data, writing of the paper, or publication decisions. The authors have nothing to declare. Trial registration number: N/A

Background: Brazil has experienced a large dengue virus (DENV) epidemic in 2019, highlighting a continual struggle with effective control and public health preparedness. Brazil is a world leader in real-time genomic surveillance of arboviruses, although such technology and expertise remains inaccessible for the vast majority of local researchers and public health workers. In 2019, we led field and classroom initiatives for the genomic surveillance of DENV in Brazil. Methods: Oxford Nanopore MinION technology was used for sequencing, focusing on generating DENV1 and DENV2 complete genomes. Using phylogenetic and epidemiological approaches conducted in real-time during a training program and subsequently through online channels, we explored the recent spatio-temporal evolution and spread of these viruses in Brazil. Findings: In the years following the Zika virus epidemic (2017-2018) reporting was at an all-time low, and significant increases in reported cases and deaths in 2019 did not reflect a higher case fatality ratio. Estimated transmission potential and reporting of other arboviruses suggests that neither arboviral reporting saturation nor climatic factors can easily explain the post-Zika period and resurgence in 2019 (respectively). Phylogenetic analysis revealed complex patterns of transmission, with lineage co-circulation and replacement, in which the North and the Southeast acted as sources of dispersion to other regions. We identified two lineages within the already reported DENV2 BR-4 clade, for which the effective reproduction number had seasonal signatures alike reported cases, with a temporal increase towards 2019 mirroring the large epidemic that year. Interpretation: We describe the recent evolution and diffusion of DENV1 and DENV2 in Brazil. Importantly, the surveillance outputs and training initiative here described serve as proof-of-concept of the potential of portable sequencing for both research and local capacity building in the area of genomic surveillance of arboviruses.

ABSTRACT Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

Summary The global emergence of Zika virus (ZIKV) in the last decade revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects such as microcephaly. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compared the transmissibility and pathogenicity of seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We found that recent African ZIKV strains largely outperformed their Asian counterparts in mosquito transmission kinetics experiments, which translated into a markedly higher epidemic potential in outbreak computer simulations. In addition, African ZIKV strains were significantly more lethal than Asian ZIKV strains in immunocompromised adult mice. Finally, prenatal infection of immunocompetent mouse embryos with an African ZIKV strain resulted in embryonic death whereas it caused microcephaly with Asian ZIKV strains. Together, our results demonstrate the high epidemic potential and pathogenicity of recent ZIKV strains from Africa. Importantly, they also imply that the African ZIKV lineage could more easily go unnoticed by public health surveillance systems than the Asian ZIKV lineage due to its propensity to cause fetal loss rather than birth defects.

Abstract Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required. Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay. Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6 – 98.2) and 100% (95% CI = 78.5 – 100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance ( = 0.913, P < 0.001). Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

Summary The human placenta is increasingly a focus of research related to early child development and the impact of maternal hyperimmune states. The ability to model human trophectoderm disease states from human pluripotent stem cells, the nature of human pluripotent stem cell potency and the mechanisms regulating human trophectoderm specification remains poorly understood. Recent work suggests that only the naive state can give rise to trophectoderm and that primed iPSC generate mixed amnionic and mesoderm lineages. Here we identify conditions that efficiently drive the specification of primed iPSC to trophectoderm, named Trophoblast Stem Cell (TSC). iPS-derived-TSC share transcriptional, morphological and functional characteristics with human in vivo cytotrophoblasts including activation of human endogenous retroviruses, expression of COVID-19 associated host factors and generation of multinucleated syncytiotrophoblasts with a large fusion index. At high densities in 5% O 2 , iPS-derived-TSC form villi-like structures and express extravillous and syncytiotrophoblast proteins HCG-β and HLA-G. Using temporal single cell RNAseq, we define the molecular changes associated with specification under three separate conditions: 1) BMP4, 2) BMP4 and inhibition of WNT, 3) activation of EGF and WNT, inhibition of TGFbeta, HDAC and ROCK signaling (named TSC). With 9,821 high-quality single cell transcriptomes, we find that BMP4 gives rise to mesenchymal cells while TS conditions lacking exogenous BMP4 generate a stable proliferating cell type that is highly similar to six week placenta cytotrophoblasts. TFAP2A programs the specification of primed iPS cells to TSC without transitioning through a naive state. TSC specification independent of exogenous BMP4 will allow for robust and reproducible studies of the cytotrophoblast component of human placenta.

Abstract The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/ ). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

Abstract Plasmablast responses and derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients. An average of 13.7% and 13.0% of plasmablast-derived IgG MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. Of thirty-two antibodies specific for the spike glycoprotein, ten recognised the receptor-binding domain (RBD), thirteen were specific for non-RBD epitopes on the S1 subunit, and nine recognised the S2 subunit. A subset of anti-spike antibodies (10 of 32) cross-reacted with other betacoronaviruses tested, five targeted the non-RBD S1, and five targeted the S2 subunit. Of the plasmablast-derived MAbs reacting with nucleocapsid, over half of them (19 of 35) cross-reacted with other betacoronaviruses tested. The cross-reactive plasmablast-derived antibodies harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. We identified 14 of 32 anti-spike MAbs that neutralised SARS-CoV-2 in independent assays at ≤ 133 nM (20 μg/ml) (five of 10 anti-RBD, three of 13 anti-non-RBD S1 subunit, six of nine anti-S2 subunit). Six of 10 anti-RBD MAbs showed evidence of blockade of ACE2 binding to RBD, and five of six of these were neutralising. Non-competing pairs of neutralising antibodies were identified, which offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.

Abstract Synthetic mRNAs are an appealing therapeutic platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison to conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining increased interest due to their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate the clinical translation of this platform. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. In this work we investigated the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response, translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented the formation of antibodies against sa-mRNA encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate drastic reduction of these dsRNA by-products upon cellulose-based purification, consequently reducing the innate immune response and improving sa-mRNA vaccination efficacy.

Antibody-antigen interaction – at antigenic local environments called B-cell epitopes – is a prominent mechanism for neutralization of infection. Effective mimicry, and display, of B-cell epitopes is key to vaccine design. Here, a physical approach is evaluated for the discovery of epitopes which evolve slowly over closely related pathogens (conserved epitopes). The approach is 1) protein flexibility-based and 2) demonstrated with the Zika virus, simulated via molecular dynamics. The approach is validated against 1) the seven structurally characterized flavivirus epitopes which have evolved the least (out of thirty-eight flavivirus-antibody structures) and 2) eight preexisting epitope and peptide discovery algorithms. For the first time, protein flexibility outperforms solvent accessible surface area as an epitope discovery metric.

BACKGROUND: A recently published randomized trial (Boulware et al., 2020, NCT04308668 ) of hydroxychloroquine (HCQ) for post-exposure prophylaxis found a reduction in Covid-19 of 17%. In the context of ambitious powering to detect a 50% reduction, this non-statistically significant finding could translate to a reduction of 22,000/130,828 cases (CDC 8/12/20) among US health care workers (HCW), impacting trajectory and resource utilization models that drive decisions on lockdowns and social distancing. Data found only in the appendix of Boulware et al. suggested greater differences in the effect of HCQ among sub-groups. There were reductions (36%) in younger ( 50 years) subjects. Our preliminary analysis revealed a significant negative correlation (slope -0.211, CI -0.328-0.094, p=0.016) between treatment lag and disease reduction, reaching 49% when initiated within one day (RR 0.51, CI 0.176-1.46, p=0.249). There were also differences in disease reduction by HCQ by type of exposure (HCW - 8% vs. household contacts - 31%; RR 0.691, CI 0.398-1.2). The definitions of exposure severity did not discriminate between the numbers or duration (> 10 minutes) of exposures. Differences between exposure types may result from younger HCW and higher risks in less trained household contacts with little access to advanced PPE. The ex-protocol use of zinc and ascorbic acid were likely confounders, as was the possibly active folate placebo. Exploratory reanalysis of the raw dataset may inform an age- and stage- nuanced approach to COVID-19 using HCQ testable by prospective studies and may provide insight into the various proposed mechanisms of HCQ. OBJECTIVES: To conduct an exploratory re-analysis of the de-identified raw dataset from a randomized study of the use of HCQ for post-exposure prophylaxis of COVID-19 with view to further defining: a) The time dependent effect of HCQ, b) The age dependent effect of HCQ; c) The sub-stratification of time- and age-dependent effects by exposure type and risk level, as well as by the use of zinc and ascorbic acid. d) The design of future clinical trials to test the hypotheses generated by this study. METHODS: Should granularity of data (by age, time-lag, level and type of exposure) be greater than that originally reported, Fisher Exact test will be used to compare the incidence of COVID-19 in HCQ- and control groups, for each sub-group stratification. Since the degree of loss of data granularity due to de-identification is yet unknown, exploratory analyses involving other demographic characteristics cannot be planned. Where sufficient data granularity exists, univariate regression analyses will be conducted to examine the effect of age- and time lag on any effect of HCQ. The possibility will be explored of conducting multivariate Cox regression analyses with propensity score matching to examine observational data relating to the use of zinc and ascorbic acid. This analysis will be expanded should a dataset from a similarly designed study (Mitja et al., 2020, NCT04304053 ), with directionally similar results, become available. This protocol was devised using the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) incorporating the WHO Trial Registration Data Set. Study Status: Protocol version 1.1, (August 19 2020) Protocol registered at: OSF Registries August 19 2020 Registration doi: https://doi.org/10.17605/OSF.IO/9RPYT

Abstract Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they are due to a direct infection of neural cells, such as neurons and astrocytes, or through indirect effects on supportive brain cells, is unknown. Here, we use brain organoids to examine SARS-CoV-2 neurotropism. We examine expression of the key viral receptor ACE2 in single-cell RNA sequencing (scRNA-seq) revealing that only a subset of choroid plexus cells but not neurons or neural progenitors express this entry factor. We then challenge organoids with both SARS-CoV-2 spike protein pseudovirus and live virus to demonstrate high viral tropism for choroid plexus epithelial cells but not stromal cells, and little to no infection of neurons or glia. We find that infected cells of the choroid plexus are an apolipoprotein and ACE2 expressing subset of epithelial barrier cells. Finally, we show that infection with live SARS-CoV-2 leads to barrier breakdown of the choroid plexus. These findings suggest that neurological complications may result from effects on the choroid plexus, an important barrier that normally prevents entry of immune cells and cytokines into the cerebrospinal fluid (CSF) and brain.

Abstract Background: In 2015-2017, the Americas experienced a highly consequencial epidemics for pregnancy and childbearing. Mainly transmitted by the mosquito Aedes aegypti , but also through sexual intercourse, the Zika virus poses the risk of congenital Zika syndrome to fetus, which includes microcephaly and other child development complications. When a public health crisis taps directly into reproductive health, typically a feminine realm, gender systems may reproduce deeply-rooted gender norms, especially those linked to cultural beliefs and expectations. This paper investigated the role of gender in two relational contexts: public health messaging to prevent Zika infections and women’s Zika and pregnancy prevention negotiation with their sexual partners during the Zika outbreak. Methods: We conducted a systematic content analysis of 94 unique pieces, governmental agencies mass-produced public health communication campaigns promoting Zika awareness. Print and online materials were collected for one year (May 2016-May 2017), such as TV ads, Internet Pop-ups, and pamphlets. We also analyzed transcripts from 16 focus groups we conducted with reproductive-aged women (18-40) in Belo Horizonte and Recife, two large cities differently affected by the Zika outbreak. Women answered open-ended questions related to personal knowledge of and experiences with the Zika virus, the related experiences of their friends and acquaintances, their primary information sources, and their perceptions of public health efforts and women’s contraceptive use. Results: Campaign pieces handling pregnancy and microcephaly demonstrated robust gendering, drastically targeting women, placing on their shoulders the responsibility for protecting a potential fetus from the disease. Campaigns neglected male´s participation on Zika prevention and contraceptive management, and failed to address Brazil´s large proportion of unplanned pregnancies. Women were placed in a double bind by being expected to prevent pregnancy and Zika amidst a relationship power dynamic that included unprotected sexual intercourse and gender norms regarding division of labor. Conclusion: Government and individual responses to the epidemics reinforced gender roles, situating pregnant women as responsible for averting mosquito bites and microcephaly, emphasizing prevention of both disease and vector among women while excluding men. Since low-socioeconomic status women possessed fewer resources to preclude infection, we also find that beyond the gender divide, this subgroup faced more pronounced Zika prevention challenges.

Abstract Poxvirus systems have been extensively used as vaccine vectors. Herein a systems vaccinology analysis of intramuscular injection sites provides detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. Adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such innate systems vaccinology approaches should inform refinements in poxvirus-based vector design.

Abstract In the last decade Open Science principles, such as Open Access, study preregistration, use of preprints, making available data and code, and open peer review, have been successfully advocated for and are being slowly adopted in many different research communities. In response to the COVID-19 pandemic many publishers and researchers have sped up their adoption of some of these Open Science practices, sometimes embracing them fully and sometimes partially or in a sub-optimal manner. In this article, we express concerns about the violation of some of the Open Science principles and its potential impact on the quality of research output. We provide evidence of the misuses of these principles at different stages of the scientific process. We call for a wider adoption of Open Science practices in the hope that this work will encourage a broader endorsement of Open Science principles and serve as a reminder that science should always be a rigorous process, reliable and transparent, especially in the context of a pandemic where research findings are being translated into practice even more rapidly. We provide all data and scripts at https://osf.io/renxy/ .

Abstract Motivation There is a need for rapid and easy to use, alignment free methods to cluster large groups of protein sequence data. Commonly used phylogenetic trees based on alignments can be used to visualize only a limited number of protein sequences. DGraph, introduced here, is a dynamic programming application developed to generate 2D-maps based on similarity scores for sequences. The program automatically calculates and graphically displays property distance (PD) scores based on physico-chemical property (PCP) similarities from an unaligned list of FASTA files. Such “PD-graphs” show the interrelatedness of the sequences, whereby clusters can reveal deeper connectivities. Results PD-Graphs generated for flavivirus (FV), enterovirus (EV), and coronavirus (CoV) sequences from complete polyproteins or individual proteins are consistent with biological data on vector types, hosts, cellular receptors and disease phenotypes. PD-graphs separate the tick- from the mosquito-borne FV, clusters viruses that infect bats, camels, seabirds and humans separately and the clusters correlate with disease phenotype. The PD method segregates the β-CoV spike proteins of SARS, SARS-CoV-2, and MERS sequences from other human pathogenic CoV, with clustering consistent with cellular receptor usage. The graphs also suggest evolutionary relationships that may be difficult to determine with conventional bootstrapping methods that require postulating an ancestral sequence. Availability and implementation DGraph is written in Java, compatible with the Java 5 runtime or newer. Source code and executable is available from the GitHub website ( https://github.com/bjmnbraun/DGraph/releases ). Documentation for installation and use of the software is available from the Readme.md file at ( https://github.com/bjmnbraun/DGraph ). Contact bjmnbraun@gmail.com or webraun@utmb.edu Supplementary information Supplementary information Table S1 and Fig. S1 are online available.

ABSTRACT Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intra-host mutation, M1404I, in the ZIKV polyprotein, located in NS2B. Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at sub-consensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I occurs rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases fitness in non-pregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to M1404, we observed that I1404 produced lower viremias in non-pregnant macaques and was a weaker competitor in tissues. In pregnant wildtype mice ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, contrasting with M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Ae. aegypti transmitted ZIKV I1404 more poorly than M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics, and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics. IMPORTANCE Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with it in non-pregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of non-pregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in non-pregnant hosts.

Abstract Melanoma differentiation-associated gene-5 (MDA5) acts as a cytoplasmic RNA sensor to detect viral dsRNA and mediates type I interferon (IFN) signaling and antiviral innate immune responses to infection by RNA viruses. Upon recognition of viral dsRNA, MDA5 is activated with K63-linked polyubiquitination and then triggers the recruitment of MAVS and activation of TBK1 and IKK, subsequently leading to IRF3 and NF-κB phosphorylation. Great numbers of symptomatic and severe infections of SARS-CoV-2 are spreading worldwide, and the poor efficacy of treatment with type I interferon and antiviral agents indicates that SARS-CoV-2 escapes from antiviral immune responses via an unknown mechanism. Here, we report that SARS-CoV-2 nonstructural protein 8 (NSP8) acts as an innate immune suppressor and inhibits type I IFN signaling to promote infection of RNA viruses. It downregulates the expression of type I IFNs, IFN-stimulated genes and proinflammatory cytokines by binding to MDA5 and impairing its K63-linked polyubiquitination. Our findings reveal that NSP8 mediates innate immune evasion during SARS-CoV-2 infection and may serve as a potential target for future therapeutics for SARS-CoV-2 infectious diseases. Importance The large-scale spread of COVID-19 is causing mass casualties worldwide, and the failure of antiviral immune treatment suggests immune evasion. It has been reported that several nonstructural proteins of severe coronaviruses suppress antiviral immune responses; however, the immune suppression mechanism of SARS-CoV-2 remains unknown. Here, we revealed that NSP8 protein of SARS-CoV-2 directly blocks the activation of the cytosolic viral dsRNA sensor MDA5 and significantly downregulates antiviral immune responses. Our study contributes to our understanding of the direct immune evasion mechanism of SARS-CoV-2 by showing that NSP8 suppresses the most upstream sensor of innate immune responses involved in the recognition of viral dsRNA.

Abstract The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2 b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.