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Prenatal resource allocation to offspring can be influenced by maternal environment and offspring value, and affect offspring survival. An important pathway for flexible maternal allocation is via egg components such as nutrients and hormones. In cooperative breeders, females with helpers may increase resource allocation to eggs—‘differential allocation’—or reduce it—‘load-lightening’. Yet, helper effects on egg composition have been poorly studied. Moreover, it is unknown how helpers’ presence modulates laying order effects on egg content and survival. Here, we investigated how maternal allocation varied with group size and laying order in the cooperatively breeding sociable weaver (Philetairus socius). We estimated interactive effects of helpers and laying order on allocation to egg mass, yolk nutrients—yolk mass, proteins, lipids, carotenoids, vitamin A and vitamin E—and hormones—testosterone, androstenedione, and corticosterone. Results concurred with the ‘differential allocation’ predictions. Females with more helpers produced later-laid eggs with heavier yolks and more lipids, and laid eggs overall richer in lipids. Proteins, antioxidants, and hormones were not found to vary with helper number. We then analyzed how helper number modulated laying order effects on survival. Females with more helpers did not specifically produce later-laid eggs with higher survival, but eggs laid by females with more helpers were overall more likely to fledge. These findings show that some egg components (yolk mass, lipids) can positively vary according to females’ breeding group size, which may improve offspring fitness.

Begging behaviour can provide information on offspring hunger levels and be used by parents to adjust food provisioning efforts. In cooperative breeders, helpers also provide care by feeding the young. However, how helpers of different sex and relatedness to the offspring respond to begging behaviour has rarely been studied in cooperatively breeding species, which limits our understanding of the indirect and/or direct benefits that helpers may obtain by responding to offspring demand. Here, we used a cooperatively breeding bird, the sociable weaver, Philetairus socius, to investigate how nest intervisit intervals of breeders and different types of helpers, distinguished by sex and relatedness, varied with acoustic begging. Moreover, we tested whether these different classes of provisioners experienced distinct levels of begging. Our results show that only breeding males, but not breeding females or helpers of any sex and relatedness to the nestlings, returned faster to the nest to feed after experiencing more begging calls. When contrasted directly, we confirmed a statistically supported difference in responses to begging between male and female breeders. Surprisingly, second-order relatives experienced more begging calls than the other classes of more related helpers and breeders. These results show that we might find differences in how provisioners respond to begging levels when classifying group members according to their potential fitness gains. In sociable weavers, the benefits and costs of adjusting feeding efforts to begging seem to differ with sex and life history stage. Experimental and more detailed investigations on begging-feeding interactions are necessary to understand the origin and prevalence of these differences across cooperatively breeding systems. (c) 2021 The Author(s). Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour. This is an open access article under the CC BY license (http://creativecommons.org/licenses/ by/4.0/).

Background Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity. Methods SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir’s dose and schedule to maximize the probability of success for COVID-19. Results Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Conclusions Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.

Cooperative actions are beneficial to the group, but presumably costly to the individual co-operators. In cooperatively breeding species, helping to raise young is thought to involve important energetic costs, which could lead to elevated exposure to reactive oxygen species, resulting in oxidative stress. However, identifying such costs can be difficult if individuals adjust their investment in helping in relation to environmental conditions or their own physiological condition. Experimental approaches are therefore required to quantify the costs of helping but, to date, these have been infrequent. Here, we combined correlational and experimental data to investigate the oxidative cost of helping-at-the-nest and how this affects helping decisions in wild sociable weavers Philetairus socius, a colonial cooperatively breeding bird. At the correlational level, we found that the probability of helping was influenced by the interaction of an individual’s oxidative state and age: compared to younger birds, older individuals were more likely to help when they had higher oxidative damage, and the opposite trend was found for younger individuals. After experimentally increasing the energetic cost of flight, manipulated helpers in breeding colonies decreased nestling feeding rates and incurred an increase in oxidative damage, which was not present in manipulated helpers in non-breeding colonies. This indicates that individuals decreased their helping behavior to minimize the associated costs. These results suggest that oxidative stress can influence helping decisions and underlie a trade-off between cooperation and self-maintenance, which is central to understanding when helping might take place in this and other species.

Influenza A virus (IAV) causes a major public health concern, because it is one of the leading causes of respiratory tract infections and hospitalization. Severe influenza has been associated with the cytokine storm, and IAV productive infection cell death in airway epithelial cells may contribute to the exacerbation of this proinflammatory event. On the other hand, IAV replication in macrophages is non-permissive and whether this immune cell may contribute to severe influenza physiopathology requires more details. Here, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We found that IAV or simply its surface glycoprotein hemagglutinin (HA) triggers necroptosis in human and murine macrophages in a Toll-like receptor-4 (TLR4) and TNF-dependent manner. Anti-TNF treatment, with the clinically approved drug etanercept, prevented the engagement of the necroptotic loop and mice mortality. impaired IAV-induced pro-inflammatory cytokine storm and lung injury. Our results implicate macrophage necroptosis with severe influenza in experimental models and potentially repurpose a clinically available therapy.Author SummaryVarious fates of cell death have an integral role in the influenza A virus (IAV) pathogenesis and lung/respiratory dysfunction. IAV physiopathology is not restricted to airway epithelial cells, where this virus actively replicated. Macrophages should support both viral clearance and priming of adaptative immune response in patients that adequately control influenza. However, during severe IAV infection, macrophages – which are unable to support a permissive viral replication - undergo disruptive cell death and contribute to the exacerbated production of proinflammatory cytokines/chemokines. We characterized this process by showing that IAV or just its surface glycoprotein hemagglutinin (HA) trigger necroptosis, a disruptive and TNF-dependent cells death. Since TNF is a hallmark of pro-inflammatory cell death, we blocked this mediator with a repurposed biomedicine etanercept, which prevented the severe IAV infection in the experimental model. The present work improves the knowledge of influenza pathophysiology by highlighting the importance of macrophage cell death during severe infection.

International audience; Climate exerts a major influence on reproductive processes, and an understanding of the mechanisms involved and which factors might mitigate adverse weather is fundamental under the ongoing climate change. Here, we study how weather and nest predation influence reproductive output in a social species, and examine whether larger group sizes can mitigate the adverse effects of these factors. We used a 7-year nest predator-exclusion experiment on an arid-region cooperatively breeding bird, the sociable weaver. We found that dry and, especially, hot weather were major drivers of nestling mortality through their influence on nest predation. However, when we experimentally excluded nest predators, these conditions were still strongly associated with nestling mortality. Group size was unimportant against nest predation and, although positively associated with reproductive success, it did not mitigate the effects of adverse weather. Hence, cooperative breeding might have a limited capacity to mitigate extreme weather effects.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1β), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Repositioning of clinical approved drugs could represent the fastest way to identify therapeutic options during public health emergencies, the majority of drugs explored for repurposing as antivirals for 2019 coronavirus disease (COVID-19) have failed to demonstrate clinical benefit. Without specific antivirals, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to cause major global mortality. Antimalarial drugs, such as chloroquine (CQ)/hydroxychloroquine (HCQ) and mefloquine have emerged as potential anti-SARS-CoV-2 antivirals. CQ/HCQ entered the Solidarity and RECOVERY clinical trials against COVID-19 and showed lack of efficacy. Importantly, mefloquine is not a 4-aminoquinoline like CQ and HCQ and has been previously repurposed for other respiratory diseases. Unlike the 4-aminoquinolines that accumulate in the high pH of intracellular lysosomes of the lung, the high respiratory tract penetration of mefloquine is driven by its high lipophilicity. While CQ and HCQ exhibit activity in Vero E6 cells, their activity is obviated in TMPRSS2-expressing cells, such as Calu-3 cells, which more accurately recapitulate in vivo entry mechanisms for SARS-CoV-2. Accordingly, here we report the anti-SARS-CoV-2 activity of mefloquine in Calu-3 type II pneumocytes and primary human monocytes. Mefloquine inhibited SARS-CoV-2 replication in Calu-3 cells with low cytotoxicity and EC50and EC90values of 1.2 and 5.3 µM, respectively. In addition, mefloquine reduced up to 68% the SARS-CoV-2 RNA levels in infected monocytes, reducing viral-induced inflammation. Mefloquine blocked early steps of the SARS-CoV-2 replicative cycle and was less prone than CQ to induce drug-associated viral mutations and synergized with RNA polymerase inhibitor. The pharmacological parameters of mefloquine are consistent with its plasma exposure in humans and its tissue-to-plasma predicted coefficient points that this drug may accumulate in the lungs. These data indicate that mefloquine could represent an orally available clinically approved drug option against COVID-19 and should not be neglected on the basis of the failure of CQ and HCQ.

Several animal models are being used to explore important features of COVID-19, nevertheless none of them recapitulates all aspects of the disease in humans. The continuous refinement and development of other options ofin vivomodels are opportune, especially ones that are carried out at BSL-2 (Biosafety Level 2) laboratories. In this study, we investigated the suitability of the intranasal infection with the murine betacoronavirus MHV-3 to recapitulate multiple aspects of the pathogenesis of COVID-19 in C57BL/6J mice. We demonstrate that MHV-3 replicated in lungs 1 day after inoculation and triggered respiratory inflammation and dysfunction. This MHV-model of infection was further applied to highlight the critical role of TNF in cytokine-mediated coronavirus pathogenesis. Blocking TNF signaling by pharmacological and genetic strategies greatly increased the survival time and reduces lung injury of MHV-3-infected mice.In vitrostudies showed that TNF blockage decreased SARS-CoV-2 replication in human epithelial lung cells and resulted in the lower release of IL-6 and IL-8 cytokines beyond TNF itself. Taken together, our results demonstrate that this model of MHV infection in mice is a useful BSL-2 screening platform for evaluating pathogenesis for human coronaviruses infections, such as COVID-19.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19., Author summary In spite of the enormous scientific efforts to understand mechanisms of SARS-CoV2-induced disease and to develop strategies to control COVID-19 pandemic, many aspects of SARS-CoV2 biology and pathogenesis remain elusive. Several RNA viruses are able to modulate the host lipid metabolism and to recruit LDs to enhance their replication/particle assembling capacity through mechanisms that vary according to the virus and the host cell infected. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are still largely unknown. Here we demonstrated that lipid droplets (LDs) participate in SARS-CoV2 infection favoring virus replication and heightening inflammatory mediator production. SARS-CoV2 infection increased the expression of key proteins in the regulation of lipid metabolism and the amounts of LDs per cell. In addition, we have found SARS-CoV2 and/or its components associated with LDs in infected cells, suggestive that LDs are recruited as part of replication compartment. Moreover, pharmacological inhibition of DGAT-1, a key enzyme for LD formation, reduces SARS-CoV2 replication, inflammatory mediator production and cell death. Our findings contribute to unveil the complex mechanism by which SARS-CoV-2 make use of cellular metabolism and organelles to coordinate different steps of the viral replication cycle and host immunity, opening new perspectives for SARS-CoV2 antiviral development.

Females may adjust prenatal allocation in relation to ecological conditions that affect reproductive success, such as weather conditions or predation risk. In cooperative breeders, helpers might also influence reproductive success, and previous studies suggest that females can lay smaller eggs or larger clutches when breeding with more helpers. Although recent work suggests that helper effects can vary according to climatic variables, how social and ecological factors interact to shape prenatal allocation is poorly understood. Here, we examine how ecological and social components of the breeding environment covary with egg mass and clutch size, using as a model the sociable weaver Philetairus socius, a colonial, cooperatively breeding passerine. The study spanned 9 years and included over 1,900 eggs from over 550 clutches. Our analyses combined natural variation in weather conditions (rainfall before each reproductive event) with a nest predator-exclusion experiment and continuous monitoring of the mother's social environment, allowing us to estimate how individual females adjust allocation to reproduction as their number of helpers varies. We found that egg mass varied consistently within females and did not clearly differ in relation to rainfall or predation risk. Contrary to previous studies, there was no evidence for plastic adjustments as females gained and lost helpers, and egg mass was instead better predicted by mother size and identity. Females laid larger clutches when breeding in environments where predation risk was experimentally reduced and after higher rainfall levels. Yet, there was no evidence for increasing clutch size as the number of helpers increased, nor for an interaction between helper effects and ecological factors. We conclude that while sociable weaver females can vary their clutch size, they show high individual consistency in egg mass. In addition, we found no evidence that females may maximize fitness through plastic prenatal allocation in relation to the number of helpers, or that the presence/absence of helper effects is modulated by rainfall levels or predation risk. These results challenge our current knowledge on some of the possible benefits of breeding with helpers and call for more long-term analyses on reproductive allocation adjustments in other cooperative systems.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with COVID-19. Here, we show that SARS-CoV-2 induces inflammasome activation and cell death by pyroptosis in human monocytes, experimentally infected and from patients under intensive care. Pyroptosis was dependent on caspase-1 engagement, prior to IL-1s production and inflammatory cell death. Monocytes exposed to SARS-CoV-2 downregulate HLA-DR, suggesting a potential limitation to orchestrate the immune response. Our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe 2019 coronavirus disease (COVID-19). Author summary Since its emergence in China in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide. Currently, the number of individuals infected with SARS-CoV-2 and in need of antiviral, anti-inflammatory, anticoagulant and more invasive treatments has overwhelmed the health systems worldwide. In our study, we found that SARS-CoV-2 is capable of inducing inflammatory cell death in human monocytes, one of the main cell types responsible for anti-SARS-CoV-2 immune response. As a consequence of this intracellular inflammatory mechanism (inflammasome engagement), an exacerbated production of inflammatory mediators occurs. The infection also decreases the expression of HLA-DR in monocytes, a molecule related to the orchestration of the immune response in case of viral infections. We also demonstrated that the HIV-1 protease inhibitor, atazanavir (ATV), prevented the uncontrolled inflammatory response, cell death and reduction in HLA-DR expression in SARS-CoV-2-infected monocytes. Our study provides relevant information on the effects of SARS-CoV-2 infection on human monocytes, as well as on the effect of ATV in preventing these pathological effects on the host.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism and energy homeostasis, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors.In vitro, SARS-CoV-2 infection modulates pathways of lipid synthesis and uptake, including CD36, SREBP-1, PPARγ and DGAT-1 in monocytes and triggered LD formation in different human cells. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected cells. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of pro-inflammatory mediators. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

Social network analyses allow studying the processes underlying the associations between individuals and the consequences of those associations. Constructing and analyzing social networks can be challenging, especially when designing new studies as researchers are confronted with decisions about how to collect data and construct networks, and the answers are not always straightforward. The current lack of guidance on building a social network for a new study system might lead researchers to try several different methods and risk generating false results arising from multiple hypotheses testing. Here, we suggest an approach for making decisions when starting social network research in a new study system that avoids the pitfall of multiple hypotheses testing. We argue that best edge definition for a network is a decision that can be made using a priori knowledge about the species and that is independent from the hypotheses that the network will ultimately be used to evaluate. We illustrate this approach with a study conducted on a colonial cooperatively breeding bird, the sociable weaver. We first identified two ways of collecting data using different numbers of feeders and three ways to define associations among birds. We then evaluated which combination of data collection and association definition maximized (a) the assortment of individuals into previously known “breeding groups” (birds that contribute toward the same nest and maintain cohesion when foraging) and (b) socially differentiated relationships (more strong and weak relationships than expected by chance). This evaluation of different methods based on a priori knowledge of the study species can be implemented in a diverse array of study systems and makes the case for using existing, biologically meaningful knowledge about a system to help navigate the myriad of methodological decisions about data collection and network inference., Determining how to build social networks for new study systems can be challenging. Pilot studies and a priori knowledge can facilitate making methodological decisions. We provide an empirical example of how to make methodological decisions by comparing different foraging networks in a colonial bird.

Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS-CoV-2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.

Current approaches of drugs repurposing against 2019 coronavirus disease (COVID-19) have not proven overwhelmingly successful and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to cause major global mortality. Daclatasvir (DCV) and sofosbuvir (SFV) are clinically approved against hepatitis C virus (HCV), with satisfactory safety profile. DCV and SFV target the HCV enzymes NS5A and NS5B, respectively. NS5A is endowed with pleotropic activities, which overlap with several proteins from SARS-CoV-2. HCV NS5B and SARS-CoV-2 nsp12 are RNA polymerases that share homology in the nucleotide uptake channel. We thus tested whether SARS-COV-2 would be susceptible these anti-HCV drugs. DCV consistently inhibited the production of infectious SARS-CoV-2 in Vero cells, in the hepatoma cell line (HuH-7) and in type II pneumocytes (Calu-3), with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than DCV, SFV and its nucleoside metabolite inhibited replication in Calu-3 cells. Moreover, SFV/DCV combination (1:0.15 ratio) inhibited SARS-CoV-2 with EC50 of 0.7:0.1 μM in Calu-3 cells. SFV and DCV prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Both drugs inhibited independent events during RNA synthesis and this was particularly the case for DCV, which also targeted secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial DCV in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Doses higher than those approved may ultimately be required, but these data provide a basis to further explore these agents as COVID-19 antiviral candidates.

Individual identification is a crucial step to answer many questions in evolutionary biology and is mostly performed by marking animals with tags. Such methods are well established but often make data collection and analyses time consuming and consequently are not suited for collecting very large datasets.Recent technological and analytical advances, such as deep learning, can help overcome these limitations by automatizing data collection and analysis. Currently one of the bottlenecks preventing the application of deep learning for individual identification is the need of hundreds to thousands of labelled pictures required for training convolutional neural networks (CNNs).Here, we describe procedures that improve data collection and allow individual identification in captive and wild birds and we apply it to three small bird species, the sociable weaver Philetairus socius, the great tit Parus major and the zebra finch Taeniopygia guttata.First, we present an automated method that allows the collection of large samples of individually labelled images. Second, we describe how to train a CNN to identify individuals. Third, we illustrate the general applicability of CNN for individual identification in animal studies by showing that the trained CNN can predict the identity of birds from images collected in contexts that differ from the ones originally used to train the CNNs. Fourth, we present a potential solution to solve the issues of new incoming individuals.Overall our work demonstrates the feasibility of applying state-of-the-art deep learning tools for individual identification of birds, both in the lab and in the wild. These techniques are made possible by our approaches that allow efficient collection of training data. The ability to conduct individual identification of birds without requiring external markers that can be visually identified by human observers represents a major advance over current methods.

The original version of this article unfortunately contained a mistake. The authors noticed an accidental duplication of Fig. 1E. The correct figure can be found below. The original article has been corrected. © 2021 The Author(s).

Zika virus (ZIKV) causes significant public health concerns because of its association with congenital malformations, neurological disorders in adults, and, more recently, death. Considering the necessity to mitigate ZIKV-associated diseases, antiviral interventions are an urgent necessity. Sofosbuvir, a drug in clinical use against hepatitis C virus (HCV), is among the FDA-approved substances endowed with anti-ZIKV activity. In this work, we further investigated the in vivo activity of sofosbuvir against ZIKV. Neonatal Swiss mice were infected with ZIKV (2 × 107 PFU) and treated with sofosbuvir at 20 mg/kg/day, a concentration compatible with pre-clinical development of this drug. We found that sofosbuvir reduced acute levels of ZIKV from 60 to 90% in different anatomical compartments, such as the blood plasma, spleen, kidney, and brain. Early treatment with sofosbuvir doubled the percentage and time of survival of ZIKV-infected animals. Sofosbuvir also prevented the acute neuromotor impairment triggered by ZIKV. In the long-term behavioural analysis of ZIKV-associated sequelae, sofosbuvir prevented loss of hippocampal- and amygdala-dependent memory. Our results indicate that sofosbuvir inhibits ZIKV replication in vivo, which is consistent with the prospective necessity of antiviral drugs to treat ZIKV-infected individuals.

Background: Neuraminidase inhibitors (NAIs) are the only class of antivirals in clinical use against influenza virus approved worldwide. However, approximately 1-3% of circulating strains present resistance mutations to oseltamivir (OST), the most used NAI. Therefore, it is important to catalogue new molecules to inhibit influenza virus, especially OST-resistant strains. Natural products from tropical plants used for human consumption represent a worthy class of substances. Their use could be stimulated in resource-limited setting where the access to expensive antiviral therapies is restricted. Methods: We evaluated the anti-influenza virus activity of agathisflavone derived from Anacardium occidentale L. Results: The neuraminidase (NA) activity of wild-type and OST-resistant influenza virus was inhibited by agathisflavone, with IC50 values ranging from 20 to 2.0 µM, respectively. Agathisflavone inhibited influenza virus replication with EC50 of 1.3 µM. Sequential passages of the virus in the presence of agathisflavone revealed the emergence of mutation R249S, A250S and R253Q in the NA gene. These changes are outside the OST binding region, meaning that agathisflavone targets this viral enzyme at a region different than conventional NAIs. Conclusion: Altogether our data suggest that agathisflavone has a promising chemical structure for the development of anti-influenza drugs.

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.

Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDβ2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDβ2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is a consolidated public health problem, in tropical and subtropical regions of the world, where control of CHIKV vector, mosquitos of theAedesgenus, failed. Since there is no vaccine or specific treatment against CHIKV, infected patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. Recently, the structure and activity of CHIKV RNA polymerase was partially resolved, revealing similar aspects with the enzyme counterparner on other positive sense RNA viruses, such as members of the Flaviviridae family. We then evaluated if sofosbuvir, clinically approved against hepatitis C virus RNA polymerase, which also aims to dengue, Zika and yellow fever viruses replication, would inhibit CHIKV replication. Indeed, sofosbuvir was 5-times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell (iPS)-derived astrocytes was less sensitive to sofosbuvir’s, compared to hepatoma cells – this drug still impaired virus production and cell death in a MOI-dependent manner. Sofosbuvir also exhibited antiviral activityin vivo, by preventing CHIKV-induced paw oedeme in adult mice, at 20 mg/kg/day, and mortality on neonate mice model, at 40 and 80 mg/kg/day. Our data demonstrates that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. Since this is a clinically approved drug, it could pave the way to become a therapeutic option against CF.

Yellow fever virus (YFV) is a member of the Flaviviridae family, that causes major mortality. In Brazil, YFV activity increased in the last years. It has been registered that sylvatic, instead of urban, yellow fever (YF) leads our contemporary public health concern. Low vaccinal coverage leaves the human population near the jangle vulnerable to the outbreak, making it necessary to identify therapeutic options. Repurposing of clinically approved antiviral drugs represents an alternative for such identification. Other Flaviviruses, such Zika (ZIKV) and dengue (DENV) viruses, are susceptible to Sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Moreover, sofosbuvir has a safety record on critically ill hepatic patients, making it an attractive option. Our data show that YFV RNA polymerase uses conserved amino acid resides for nucleotide binding to dock sofosbuvir. This drug inhibited YFV replication in different lineages of human hepatoma cells, Huh-7 and HepG2, with EC50 value of 4.8 µM. Sofosbuvir protected YFV-infected neonatal Swiss mice from mortality and weight loss. Our pre-clinical results indicate that sofosbuvir could represent an option against YFV.

Scientific Reports 7: Article number: 40920; published online 18 January 2017; updated on 24 April 2017 The Competing Financial Interests section in this Article is incorrect and should read: “Dr. Karin Brüning is a member of the BMK consortium, able to produce sofosbuvir”.

Zika virus (ZIKV) is a member of the Flaviviridae family, along with other agents of clinical significance such as dengue (DENV) and hepatitis C (HCV) viruses. Since ZIKV causes neurological disorders during fetal development and in adulthood, antiviral drugs are necessary. Sofosbuvir is clinically approved for use against HCV and targets the protein that is most conserved among the members of the Flaviviridae family, the viral RNA polymerase. Indeed, we found that sofosbuvir inhibits ZIKV RNA polymerase, targeting conserved amino acid residues. Sofosbuvir inhibited ZIKV replication in different cellular systems, such as hepatoma (Huh-7) cells, neuroblastoma (SH-Sy5y) cells, neural stem cells (NSC) and brain organoids. In addition to the direct inhibition of the viral RNA polymerase, we observed that sofosbuvir also induced an increase in A-to-G mutations in the viral genome. Together, our data highlight a potential secondary use of sofosbuvir, an anti-HCV drug, against ZIKV.

Yellow fever virus (YFV) is a member of the Flaviviridae family. In Brazil, yellow fever (YF) cases have increased dramatically in sylvatic areas neighboring urban zones in the last few years. Because of the high lethality rates associated with infection and absence of any antiviral treatments, it is essential to identify therapeutic options to respond to YFV outbreaks. Repurposing of clinically approved drugs represents the fastest alternative to discover antivirals for public health emergencies. Other Flaviviruses, such as Zika (ZIKV) and dengue (DENV) viruses, are susceptible to sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Our data showed that sofosbuvir docks onto YFV RNA polymerase using conserved amino acid residues for nucleotide binding. This drug inhibited the replication of both vaccine and wild-type strains of YFV on human hepatoma cells, with EC50 values around 5 μM. Sofosbuvir protected YFV-infected neonatal Swiss mice and adult type I interferon receptor knockout mice (A129-/-) from mortality and weight loss. Because of its safety profile in humans and significant antiviral effects in vitro and in mice, Sofosbuvir may represent a novel therapeutic option for the treatment of YF. Key-words: Yellow fever virus; Yellow fever, antiviral; sofosbuvir, Author summary Yellow fever virus is transmitted by mosquitoes and its infection may be asymptomatic or lead to a wide clinical spectrum ranging from a mild febrile illness to a potentially lethal viral hemorrhagic fever characterized by liver damage. Although a yellow fever vaccine is available, low coverage allows 80,000–200,000 cases and 30,000–60,000 deaths annually worldwide. There are no specific therapy and treatment relies on supportive care, reinforcing an urgent need for antiviral repourposing. Here, we showed that sofosbuvir, clinically approved against hepatitis C, inhibits yellow fever virus replication in liver cell lines and animal models. In vitro, sofosbuvir inhibits viral RNA replication, decreases the number of infected cells and the production of infectious virus particles. These data is particularly relevante since the liver is the main target of yellow fever infection. Sofosbuvir also protected infected animals from mortality, weight loss and liver injury, especially prophylatically. Our pre-clinical results supports a second use of sofosbuvir against yellow fever.

SummaryZika virus (ZIKV) is a member of Flaviviridae family, as other agents of clinical significance, such as dengue (DENV) and hepatitis C (HCV) viruses. ZIKV spread from Africa to Pacific and South American territories, emerging as an etiological pathogen of neurological disorders, during fetal development and in adulthood. Therefore, antiviral drugs able to inhibit ZIKV replication are necessary. Broad spectrum antivirals, such as interferon, ribavirin and favipiravir, are harmful for pregnant animal models and women. The clinically approved uridine nucleotide analog anti-HCV drug, sofosbuvir, has not been affiliated to teratogenicity. Sofosbuvir target the most conserved protein over the members of the Flaviviridae family, the viral RNA polymerase. We thus studied ZIKV susceptibility to sofosbovir. We initially characterized a Brazilian ZIKV strain for use in experimental assays. Sofosbuvir inhibits the Brazilian ZIKV replication in a dose-dependent manner, both in BHK-21 cells and SH-Sy5y, by targeting ZIKV RNA polymerase activity, with the involvement of conserved amino acid residues over the members of Flaviviridae family. The identification of clinically approved antiviral drugs endowed with anti-ZIKV could reduce the time frame in pre-clinical development. Altogether, our data indicates that sofosbuvir chemical structure is endowed with anti-ZIKV activity.

Cobalt–iron magnetic composites prepared by the thermal treatment of an iron oxide-rich soil in the presence of sucrose and cobalt(II) sulfate are efficient heterogeneous catalysts for the liquid-phase aerobic oxidation of thiols into disulfides. The materials have been characterized by Mossbauer spectroscopy, XRD, N 2 adsorption–desorption, and elemental analysis. It has been shown that the isomorphic substitution of iron by cobalt occurs preferentially in the framework of the wustite (FeO) phase and strongly affects the catalytic behavior of the material. The choice of a solvent is critically important for the efficiency of the reaction. In weakly basic solvents, such as dimethylformamide and dimethylacetamide, disulfides can be obtained in near-quantitative yields at low catalyst loading (0.008 mol%). A significant practical advantage of this environment-friendly process is the use of inexpensive magnetically recoverable materials as catalysts and oxygen as a final oxidant as well as mild alkali free conditions. Of particular note are the stability of the catalyst toward leaching and the possibility of catalyst recycling without any special treatment.

Background Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a potentially lethal complication of clinical malaria. Acute lung injury in MA-ARDS shares features with ARDS triggered by other causes, including alveolar inflammation and increased alveolar-capillary permeability, leading to leak of protein-rich pulmonary oedema fluid. Mechanisms and physiologic alterations in MA-ARDS can be examined in murine models of this syndrome. Integrin αDβ2 is a member of the leukocyte, or β2 (CD18), sub-family of integrins, and emerging observations indicate that it has important activities in leukocyte adhesion, accumulation and signalling. The goal was to perform analysis of the lungs of mice wild type C57Bl/6 (aD+/+) and Knockout C57Bl/6 (aD−/−) with malaria-associated acute lung injury to better determine the relevancy of the murine models and investigate the mechanism of disease. Methods C57BL/6 wild type (aD+/+) and deficient for CD11d sub-unit (aD−/−) mice were monitored after infection with 105Plasmodium berghei ANKA. CD11d subunit expression RNA was measured by real-time polymerase chain reaction, vascular barrier integrity by Evans blue dye (EBD) exclusion and cytokines by ELISA. Protein and leukocytes were measured in bronchoalveolar lavage fluid (BALF) samples. Tissue cellularity was measured by the point-counting technique, F4/80 and VCAM-1 expression by immunohistochemistry. Respiratory function was analysed by non-invasive BUXCO and mechanical ventilation. Results Alveolar inflammation, vascular and interstitial accumulation of monocytes and macrophages, and disrupted alveolar-capillary barrier function with exudation of protein-rich pulmonary oedema fluid were present in P. berghei-infected wild type mice and were improved in αDβ2-deficient animals. Key pro-inflammatory cytokines were also decreased in lung tissue from αD−/− mice, providing a mechanistic explanation for reduced alveolar-capillary inflammation and leak. Conclusions The results indicate that αDβ2 is an important inflammatory effector molecule in P. berghei-induced MA-ARDS, and that leukocyte integrins regulate critical inflammatory and pathophysiologic events in this model of complicated malaria. Genetic deletion of integrin subunit αD in mice, leading to deficiency of integrin αDβ2, alters lung inflammation and acute lung injury in a mouse model of MA-ARDS caused by P. berghei.