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Identification of host determinants of coronavirus infection informs mechanisms of viral pathogenesis and can provide new drug targets. Here we demonstrate that mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) chromatin remodeling complexes, specifically canonical BRG1/BRM-associated factor (cBAF) complexes, promote severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and represent host-directed therapeutic targets. The catalytic activity of SMARCA4 is required for mSWI/SNF-driven chromatin accessibility at the ACE2 locus, ACE2 expression and virus susceptibility. The transcription factors HNF1A/B interact with and recruit mSWI/SNF complexes to ACE2 enhancers, which contain high HNF1A motif density. Notably, small-molecule mSWI/SNF ATPase inhibitors or degraders abrogate angiotensin-converting enzyme 2 (ACE2) expression and confer resistance to SARS-CoV-2 variants and a remdesivir-resistant virus in three cell lines and three primary human cell types, including airway epithelial cells, by up to 5 logs. These data highlight the role of mSWI/SNF complex activities in conferring SARS-CoV-2 susceptibility and identify a potential class of broad-acting antivirals to combat emerging coronaviruses and drug-resistant variants.

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic—Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats’ unique traits.

International audience; Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities—rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.

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

Zika virus (ZIKV) infection is a major public health threat, making the study of its biology a matter of great importance. By analyzing the viral-host protein interactions and proposing them as new drug targets, we would diminish the emergence of new resistant strains. In this work, we have shown that human cytoplasmic dynein-1 (Dyn) interacts with the ZIKV. We additionally demonstrate that the envelope protein of the ZIKV and the dimerization domain of the heavy chain of Dyn binds directly without dynactin or cargo adaptor. In addition, we have analyzed this interaction in Vero cells, where we are proposing that the interaction ZIKV-Dyn is finely tuned within the replication cycle. Altogether, our data suggest a new step in the previously described replication cycle of the ZIKV, introducing a suitable molecular target to modulate infection by ZIKV.

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6—all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants., Nature, 602 (7897), ISSN:0028-0836, ISSN:1476-4687

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

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. Few examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on Golgi complex imposed by virion and NS1 processing and secretion.

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated sixteen of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.

Hurting the virus by targeting the host Many host proteins play a role in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and some are required for viral replication and translation. There are efforts toward finding drugs that target viral proteins, but a complementary approach is to target these required host proteins. White et al. explored the antiviral activity of the cyclic depsipeptide drug plitidepsin, which targets the hosts cell's translational machinery (see the Perspective by Wong and Damania). The authors show that in cells, the drug is substantially more potent than remdesivir against SARS-CoV-2, with limited cellular toxicity. Prophylactic treatment protected mice against SARS-CoV-2 infection, so further investigation of plitidepsin as a therapeutic is warranted. Science, this issue p. 926; see also p. 884, Plitidepsin is a host-targeted antiviral against SARS-CoV-2, with potent efficacy both in vitro and in mouse models., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Plitidepsin is a marine-derived cyclic-peptide that inhibits SARS-CoV-2 replication at low nanomolar concentrations by the targeting of host protein eEF1A (eukaryotic translation-elongation-factor-1A). We evaluated a model of intervention with plitidepsin in hospitalized COVID-19 adult patients where three doses were assessed (1.5, 2 and 2.5 mg/day for 3 days, as a 90-minute intravenous infusion) in 45 patients (15 per dose-cohort). Treatment was well tolerated, with only two Grade 3 treatment-related adverse events observed (hypersensitivity and diarrhea). The discharge rates by Days 8 and 15 were 56.8% and 81.8%, respectively, with data sustaining dose-effect. A mean 4.2 log10 viral load reduction was attained by Day 15. Improvement in inflammation markers was also noted in a seemingly dose-dependent manner. These results suggest that plitidepsin impacts the outcome of patients with COVID-19., This study has been funded by Pharmamar, S.A. (Madrid, Spain). This work was supported by grants from the Government of Spain (PIE_INTRAMURAL_ LINEA 1 - 202020E079; PIE_INTRAMURAL_CSIC-202020E043). The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols pharmaceutical. We also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). N.I.U. has non-restrictive funding from PharmaMar to study the antiviral effect of Plitidepsin. N.J.K. was funded by grants from the National Institutes of Health (P50AI150476, U19AI135990, U19AI135972, R01AI143292, R01AI120694, and P01AI063302); by the Excellence in Research Award (ERA) from the Laboratory for Genomics Research (LGR), a collaboration between UCSF, UCB, and GSK (#133122P); by the Roddenberry Foundation, and gifts from QCRG philanthropic donors. This work was supported by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement #HR0011-19-2-0020. The views, opinions, and/or findings contained in this material are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. This research was partly funded by CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grants U19AI142733, U19AI135972 and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to AG-S. S.Y. received funding from a Swiss National Foundation (SNF) Early Postdoc Mobility fellowship (P2GEP3_184202).

SummaryEffective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS- CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold.HighlightsSeveral HCV protease inhibitors are predicted to inhibit SARS-CoV-2 Mpro and PLpro.Seven HCV drugs inhibit Mpro enzyme activity, four HCV drugs inhibit PLpro.Seven HCV drugs inhibit SARS-CoV-2 replication in Vero and/or human cells.HCV drugs simeprevir and grazoprevir synergize with remdesivir to inhibit SARS- CoV-2.eTOC blurbBafna, White and colleagues report that several available hepatitis C virus drugs inhibit the SARS-CoV-2 Mpro and/or PLpro proteases and SARS-CoV-2 replication in cell culture. Two drugs, simeprevir and grazoprevir, synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir antiviral activity as much as 10-fold.Abstract Figure

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID., Graphical Abstract, Jansen, Reimer, Nagai et al report that SARS-CoV-2 infects kidney cells and is associated with kidney fibrosis in patients. Using single cell transcriptomics of infected kidney organoids, they show that SARS-CoV-2 causes kidney injury and stimulates pro-fibrotic signaling. Viral infection in organoids was inhibited by a recently developed protease blocker.

Flaviviruses of major medical importance worldwide such as dengue (DENV), Zika (ZIKV), and yellow fever (YFV) viruses are transmitted by mosquitoesAedes sp. The non-structural protein 1 (NS1) of these flaviviruses is secreted from the infected cells using different secretion routes depending on the cell and virus nature. The NS1 of DENV and ZIKV contain in the hydrophobic region a conserved caveolin binding domain (CBD) (ΦXXΦXXXXΦ), which is not conserved in YFV NS1. To ascertain the role of the CBD in the secretory route followed by flavivirus NS1, expression vectors for the NS1 of DENV2, ZIKV and YFV were constructed. Using site-directed mutagenesis, substitutions were made in the aromatic residues within CBD; in addition, the full domain was replaced by those of other flaviviruses, creating chimeras in the CBD of NS1. Substitutions of the aromatic residues to Ala or Thr, or CBD chimeras, results in increased sensitivity of NS1 secretion to brefeldin A treatment, indicating a change to a classical secretion pathway. Likewise, the insertion of the DENV/ZIKV CBD into the recombinantGaussia-Luciferase results in a loss of sensitivity to BFA treatment, in luciferase secretion. These results suggest that the CBD sequence is a molecular determinant for the unconventional secretory route followed by DENV and ZIKV NS1 in mosquito cells. However, the cellular components that recognize the CBD in the NS1 of DENV and ZIKV and redirect them to an unconventional route and if this secretion route confers unique functions to NS1 within the vector mosquito are aspects currently unknown.ImportanceFlaviviruses are an important cause of mosquito borne diseases to humans. We have previously demonstrated that the non-structural protein 1 from dengue and zika virus are secreted efficiently from mosquito cells using an unconventional route, that depends on caveolin and molecular chaperones. In this work, we show evidence indicating that a caveolin binding domain, well conserved and exposed in dengue and Zika virus NS1, but absent in other flaviviruses such as yellow fever virus or West Nile virus, is important in determining the unconventional secretion pathway followed by dengue and zika virus NS1 in mosquito cells. The unique secretory pathway followed by NS1 in mosquito cells may result in distinctive viral-cellular protein associations required to facilitate viral infection in the mosquito vector. To identify viral and cellular elements that could disturb the traffic of dengue and Zika virus NS1 may be important to design of strategies for vector control.

The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed ten hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum was resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir’s antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir., Graphical Abstract, Bafna et al. report that several available hepatitis C virus drugs inhibit the SARS-CoV-2 Mpro and/or PLpro proteases, and SARS-CoV-2 replication in cell culture. The four HCV drugs that inhibit PLpro enzyme activity also synergize with remdesivir to inhibit virus replication, increasing remdesivir and HCV drugs antiviral activity.

Dengue virus (DENV) is a mosquito-borne virus of the familyFlaviviridae.The RNA viral genome encodes for a polyprotein that is co-translationally processed into three structural proteins and seven non-structural proteins. The non-structural protein 1 (NS1) is a multifunctional viral protein actively secreted in vertebrate and mosquito cells during DENV infection. In mosquito cells, NS1 is secreted in a caveolin-1 (CAV-1) dependent manner by an unconventional pathway. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40 and CyA which is responsible for cholesterol traffic inside the cell. In this work, we demonstrate that in infected mosquito cells, DENV NS1 is secreted by an early and unconventional route that bypasses the Golgi apparatus in close association with the CCC. Treatment of mosquito cells with classic secretion inhibitors such as brefeldin A, golgicide A and Fli-06 showed no effect on NS1 secretion, but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV1, FKBP52 with siRNAs or the inhibition of CyA by cyclosporine A resulted in significant decrease in NS1 secretion without affecting virion release. Using co-localization, co-inmunoprecipitation and proximity ligation assays, NS1 was found to co-localize and interact with all the protein components of the CCC in mosquito infected cells. In addition, CAV-1 and FKBP52 expression was found augmented in DENV infected cells. Finally, the treatment of ZIKV infected mosquito cells with brefeldin A and golgicide A showed no effect on NS1 secretion, while affecting virion release. ZIKV NS1 was also found to co-localize with CAV-1 in infected mosquito cells. These results suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway observed for DENV NS1. The association of NS1 with the cholesterol transporter CCC agrees with the lipoprotein nature of secreted hexameric NS1.AUTHOR SUMMARYDengue protein NS1 is secreted in infected mosquito and vertebrate cells. In humans, secreted NS1 have been associated with pathogenesis. In mosquito cells, NS1 follows an unconventional secretion pathway that is dependent on Caveolin-1. This work shows that in mosquito cells, NS1 secretion is associated to the chaperone caveolin complex, a complex formed by caveolin-1 and several chaperones, in charge of cholesterol transport within the cells. Reduction of the expression or the activity of chaperone caveolin complex in mosquito infected cells, diminished the secretion of NS1 without affecting virion release. Direct interaction between NS1 and the chaperone caveolin complex proteins was demonstrated by several assays. Moreover, increased expression of the caveolin-1 and co-chaperone FKBP52 during dengue infection was found, presumably in response to the higher requirements of these proteins during dengue virus infection. Results obtained with ZIKV infected mosquito cells suggest that also ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. The functions of secreted NS1 within mosquito are unclear. However, giving the importance of the soluble NS1 in the vertebrate host, manipulation of the NS1 secretory route may prove a valuable strategy for dengue mosquito control and patient treatment.

[Background] Parasites can exert selection pressure on their hosts through effects on survival, on reproductive success, on sexually selected ornament, with important ecological and evolutionary consequences, such as changes in population viability. Consequently, hemoparasites have become the focus of recent avian studies. Infection varies significantly among taxa. Various factors might explain the differences in infection among taxa, including habitat, climate, host density, the presence of vectors, life history and immune defence. Feeding behaviour can also be relevant both through increased exposure to vectors and consumption of secondary metabolites with preventative or therapeutic effects that can reduce parasite load. However, the latter has been little investigated. Psittaciformes (parrots and cockatoos) are a good model to investigate these topics, as they are known to use biological control against ectoparasites and to feed on toxic food. We investigated the presence of avian malaria parasites (Plasmodium), intracellular haemosporidians (Haemoproteus, Leucocytozoon), unicellular flagellate protozoans (Trypanosoma) and microfilariae in 19 Psittaciformes species from a range of habitats in the Indo-Malayan, Australasian and Neotropical regions. We gathered additional data on hemoparasites in wild Psittaciformes from the literature. We considered factors that may control the presence of hemoparasites in the Psittaciformes, compiling information on diet, habitat, and climate. Furthermore, we investigated the role of diet in providing antiparasitic secondary metabolites that could be used as self-medication to reduce parasite load., [Results] We found hemoparasites in only two of 19 species sampled. Among them, all species that consume at least one food item known for its secondary metabolites with antimalarial, trypanocidal or general antiparasitic properties, were free from hemoparasites. In contrast, the infected parrots do not consume food items with antimalarial or even general antiparasitic properties. We found that the two infected species in this study consumed omnivorous diets. When we combined our data with data from studies previously investigating blood parasites in wild parrots, the positive relationship between omnivorous diets and hemoparasite infestation was confirmed. Individuals from open habitats were less infected than those from forests., [Conclusions] The consumption of food items known for their secondary metabolites with antimalarial, trypanocidal or general antiparasitic properties, as well as the higher proportion of infected species among omnivorous parrots, could explain the low prevalence of hemoparasites reported in many vertebrates., Laboratory work was supported by the SYNTHESYS Project (ES-TAF 4110 and ES-TAF 4542; http://www.synthesys.info/), financed by the European Community Research Infrastructure Action under the FP7 “Capacities” Programme at the Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain. The extensive fieldwork was carried out with financial support from different organizations. For population number see Table 1. Organizations and grant numbers (if available): 1 (Katala Foundation); 2, 3, 4, 5, 10 and 11 (Loro Parque Fundación (Spain), Polish Ministry of Science and Higher Education (Grant 2P04F 001 29), Conservation des Espèces et Populations Animales (France), La Fondation Nature et Découvertes (France), Fonds für bedrohte Papageien - Zoologische Gesellschaft für Arten und Populationsschutz (Germany); 6, 7, 8 and 9 (New Zealand Department of Conservation, Institute of Natural Sciences (Massey University), Motuihe Island Trust, Landcare Research and National Council of Science from Mexico (CONACYT)); 12, 13 and 14 (World Parrot Trust); 15, 24 and 25 (PIP 112-20150100598 CONICET, PICT 2015-2281 ANPCyT, Argentina, and World Parrot Trust); 16 (Projects 657 and 1365, Instituto Venezolano de Investigaciones Científicas, Venezuela); 17 and 25 (Fundação Neotropica do Brasil, Brazil); 18 and 19 (the City Council of Viedma Río Negro, Argentina, World Parrot Trust, and PQ was supported by the Deutsche Forschungsgemeinschaft (DFG) (Qu 148/1 ff.)); 20 (FONDECYT 1140548, ICM-P05-002, and PFB-23-CONICYT, Chile, to RAV, and Ministerio de Economía y Competitividad and European Regional Development Fund (MINECO/FEDER) CGL2015-67789-C2-1-P / BOS to SM); 21 (PICT 2012-2926 ANPCyT); 22 and 23 (Sociedade de Pesquisa em Vida Selvagem e Educação Ambiental and CEMAVE, Brazil).

The role of Ca

Trypanosoma evansi is a mammal generalist protozoon which causes negative effects on health and productivity in bovine and equine herds in South America, Europe, Asia and Africa. By molecular methods, we screened the presence of that parasite together with other trypanosome species in 105 bats of 10 species collected in arid zones of northern Venezuela. The first molecular approach was fluorescent fragment length barcoding (FFLB), which relies on amplification of relative small regions of rRNA genes (four loci) and fluorescence detection. By FFLB, 17 samples showed patterns of possible trypanosomatid infections. These samples were used to test presence of trypanosomes by PCR using the following DNA markers: V7–V8 SSU rRNA, gGAPDH and kDNA minicircle regions. Only in one individual of the nectar-feeding bat, Leptonycteris curasoae, we were able to amplify 1000 bp of the trypanosome kDNA minicircle. That PCR product was sequenced and the parasite species was determined by NCBI-BLAST and phylogenetic analysis. Both analyses showed that the minicircle sequence corresponds to Trypanosoma evansi. The phylogenetic analysis of the sequence obtained in this study clustered with a T. evansi sequence obtained in a Venezuelan capybara, Hydrochoerus hydrochaeris, and distant of others two T. evansi sequences obtained in a Colombian capybara and horse. This result supports the hypothesis of multiple origins of T. evansi in South America.

Avian hemosporidian parasites of the genera Haemoproteus, Plasmodium, and Leucocytozoon are transmitted by different dipteran vectors. In the present work, we looked for the presence of these parasites in 47 birds from 12 families, which were sampled in the migratory corridor Paso de Portachuelo, located at the Henri Pittier National Park, Venezuela. The presence of the parasites was evidenced by amplification of a region of 471 bp of their cytochrome b gene. This region of the marker presents enough polymorphism to identify most of the mitochondrial lineages. Therefore, the obtained amplicons were sequenced, not only to identify the genus of the parasites sampled, but also to analyze their genetic diversity in the study area. The overall parasite prevalence was low (11%). We reported, for the first time, Plasmodium in birds of the species Formicarius analis and Chamaeza campanisona (Formicariidae) and Haemoproteus in Geotrygon linearis (Columbidae). A phylogenetic tree was generated using the Haemoproteus, Plasmodium, and Leucocytozoon sequences obtained in this study, together with representative sequences from previous studies. The highest genetic diversities between the two Haemoproteus lineages (11.70%) and among the three Plasmodium lineages (7.86%) found in this study are also similar to those found when lineages reported in the literature were used. These results indicate that in the migratory corridor Paso de Portachuleo, representative parasite lineages are found, making this location an attractive location for future studies.

The focus of this study was the detection of equine piroplasmosis in Distrito Capital, Miranda, Aragua, Guarico and Apure States from Venezuela, using two methods: Competitive-Inhibition ELISA and multiplex PCR and the analysis of the possible differences in occurrence in relation to the primary purpose of the horses, which is related to varied degrees of exposure to tick. Antibody levels to Babesia caballi and Theileria equi were assessed in 694 equine serum samples using Competitive-Inhibition ELISA, while PCR assays were performed in 136 horses, using two sets of oligonucleotides to establish the presence of T. equi, B. caballi or both. The overall seroprevalence of equine piroplasmosis was 50.2%, antibodies to B. caballi were found in 161 horses (23.2%), whereas 97 (14.0%) were seropositive to T. equi and 90 (13.0%) were positives to both parasites (mixed infections). PCR determinations (n=136) showed a prevalence of 66.2%, distributed in 84 (61.8% positives) for T. equi and, 6 (4.4%) were positive to both parasites. The cELISA showed higher levels of prevalence of B. caballi and mixed infections, as compared to the PCR method. This discrepancy can be explained by the different parameters that are evaluated by each technique, PCR detect the parasite itself, while cELISA detects antibodies to the parasite. By PCR, the highest prevalence was found in Apure state, where 92.3% of the samples were positive to T. equi infections. In this locality, free grazing animals are used for livestock management. This high prevalence may be linked to the tick species present in that area. More epidemiological studies will be necessary to assess the epidemiological status of equine piroplasmosis in Venezuela.

By amplifying and sequencing a mitochondrial DNA barcode (cytb), we screened the frequency of haemosporidian parasites (Haemoproteus, Plasmodium, Leucocytozoon) in 366 birds (23 species) from Margarita and Coche islands, Venezuela. In Coche (N = 24), none of the birds were infected, while in Margarita (N = 342) 13 individuals were (3.8%). The frequency of these parasites in endemic bird subspecies was more than double than in non-endemic forms (endemics = 7.3% vs non-endemic = 3.1%, N = 342), but it was not statistically significant. We found eight parasite lineages, four of which are novel (COLPAS09, COLSQU03, COLSQU04, HYPRUF01). COLSQU03, recorded in a dove (Columbina squammata), falls within the Haemoproteus (Parahaemoproteus) subgenus. This was unexpected because a strong signal of co-speciation has been found between Columbidae and the Haemoproteus (Haemoproteus) subgenus, suggesting a host-switching event.