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Recent studies have confirmed that lung microvascular endothelial injury plays a critical role in the pathophysiology of COVID-19. Our group and others have demonstrated the beneficial effects of H2S in several pathological processes and provided a rationale for considering the therapeutic implications of H2S in COVID-19 therapy. Here, we evaluated the effect of the slow-releasing H2S donor, GYY4137, on the barrier function of a lung endothelial cell monolayer in vitro, after challenging the cells with plasma samples from COVID-19 patients or inactivated SARS-CoV-2 virus. We also assessed how the cytokine/chemokine profile of patients’ plasma, endothelial barrier permeability, and disease severity correlated with each other. Alterations in barrier permeability after treatments with patient plasma, inactivated virus, and GYY4137 were monitored and assessed by electrical impedance measurements in real time. We present evidence that GYY4137 treatment reduced endothelial barrier permeability after plasma challenge and completely reversed the endothelial barrier disruption caused by inactivated SARS-CoV-2 virus. We also showed that disease severity correlated with the cytokine/chemokine profile of the plasma but not with barrier permeability changes in our assay. Overall, these data demonstrate that treatment with H2S-releasing compounds has the potential to ameliorate SARS-CoV-2-associated lung endothelial barrier disruption.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East that can affect humans and ruminant livestock. Currently, there are no approved vaccines or therapeutics for the treatment of severe RVF disease in humans. Tilorone-dihydrochloride (Tilorone) is a broad-spectrum antiviral candidate that has previously shown efficacy against a wide range of DNA and RNA viruses, and which is clinically utilized for the treatment of respiratory infections in Russia and other Eastern European countries. Here, we evaluated the antiviral activity of Tilorone against Rift Valley fever virus (RVFV). In vitro, Tilorone inhibited both vaccine (MP-12) and virulent (ZH501) strains of RVFV at low micromolar concentrations. In the mouse model, treatment with Tilorone significantly improved survival outcomes in BALB/c mice challenged with a lethal dose of RVFV ZH501. Treatment with 30 mg/kg/day resulted in 80% survival when administered immediately after infection. In post-exposure prophylaxis, Tilorone resulted in 30% survival at one day after infection when administered at 45 mg/kg/day. These findings demonstrate that Tilorone has potent antiviral efficacy against RVFV infection in vitro and in vivo and supports further development of Tilorone as a potential antiviral therapeutic for treatment of RVFV infection.

The unprecedented magnitude of the 2013-2016 Ebola virus (EBOV) epidemic in West Africa resulted in over 11,000 deaths and spurred an international public health emergency. A second outbreak in 2018-2020 in DRC resulted in an additional >3400 cases and nearly 2300 deaths (WHO Disease Outbreak News: Update 26 June, 2020). These large outbreaks across geographically diverse regions highlight the need for the development of effective oral therapeutic agents that can be easily distributed for self-administration to populations with active disease or at risk of infection. Herein, we report the in vivo efficacy ofN4-hydroxycytidine (EIDD-1931), a broadly active ribonucleoside analog and the active metabolite of the prodrug EIDD-2801 (molnupiravir), in murine models of lethal EBOV infection. Twice daily oral dosing with EIDD-1931 at 200 mg/kg for 7 days, initiated either with a prophylactic dose 2 hours before infection, or as therapeutic treatment starting 6 hours post-infection, resulted in 92-100% survival of mice challenged with lethal doses of EBOV, reduced clinical signs of Ebola virus disease (EVD), reduced serum virus titers, and facilitated weight loss recovery. These results support further investigation of molnupiravir as a potential therapeutic or prophylactic treatment for EVD.

The unprecedented magnitude of the 2013-2016 Ebola virus (EBOV) epidemic in West Africa resulted in over 11 000 deaths and spurred an international public health emergency. A second outbreak in 2018-2020 in DRC resulted in an additional3400 cases and nearly 2300 deaths (WHO, 2020). These large outbreaks across geographically diverse regions highlight the need for the development of effective oral therapeutic agents that can be easily distributed for self-administration to populations with active disease or at risk of infection. Herein, we report the in vivo efficacy of N

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.

The common marmoset (Callithrix jacchus) has, in recent years, received more recognition as an ideal non-human primate (NHP) model for studies at high-biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets. Four marmosets were infected via the intranasal and intratracheal route and monitored for disease development. All four subjects developed fatal disease between days 8 and 11 post infection, with three animals showing severe respiratory disease and one marmoset recapitulating the neurologic clinical symptoms seen in humans, as well as cardiomyopathy on gross pathology. We obtained histopathological data, quantified genome copies on >25 tissue-types, and performed RNA-seq on six different organs from all infected and control marmosets. Three out of four marmosets showed pulmonary edema and hemorrhage as well as multi-focal hemorrhagic lymphadenopathy. In all animals, syncytia were evident in endothelial cells in pulmonary vessels, cells in alveolar septum, and in splenic follicles or red pulp. To define the organ-specific innate and inflammatory responses, we performed RNA-seq on six different organs from all infected and compared to naive non-infected marmoset tissues. RNA-seq gave 17.4 million reads per sample on average, with the most highly infected tissue sample, the lung of one marmoset, containing >180,000 virus-specific reads. NiV V and W transcripts comprised ~40% of all phosphoprotein-derived transcripts with V and W being very close to each other proportionally. Principal component analysis showed that in brain stem, the marmoset exhibiting neurological symptoms displayed a unique RNA transcriptome relative even to other infected marmosets. Upregulated genes in the various tissues belonged to distinct GO pathways. Additionally, one male and female animal had detectable viral reads in their ovaries (27,120) and testes (858). Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.

Over the past 20 years, Nipah virus (NiV) has emerged as a significant, highly pathogenic bat-borne paramyxovirus causing severe respiratory disease and encephalitis in humans, and human-to-human transmission has been demonstrated in multiple outbreaks. In addition to causing serious illness in humans, NiV is a zoonotic pathogen capable of infecting a wide range of other mammalian species, including pigs and horses. While NiV has caused less than 700 human cases since its discovery in 1998/1999, the involvement of intermediate agricultural hosts can result in significant economic consequences. Owing to the severity of disease, capacity for human-to-human transmission, zoonotic potential, and lack of available approved therapeutic treatment options, NiV has been listed by the World Health Organization in their Blueprint list of priority pathogens as one of the eight most dangerous pathogens to monitor and prepare countermeasures to prevent a pandemic. Here, we discuss progress towards the development of therapeutic measures for the treatment of NiV infection and disease.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines., Author summary Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever, which is primarily endemic in African countries. The virus infects a wide variety of mammalian species, including ruminants and humans, and causes devastating effects on public health and the livestock industry. Because RVFV is transmitted by several mosquito species that are ubiquitous in non-endemic areas, there is a potential risk that the virus will spread outside of the current endemic areas. However, there are no effective therapeutics nor commercially available vaccines for human use. RVFV encodes a 78kDa protein (P78) of unidentified biological function in mammalian cells. We found that a mutant RVFV expressing a higher level of P78 (P78-High virus) showed lower infectivity and less efficient replication than parental RVFV in macrophage cell lines. P78-High virus and a mutant RVFV lacking P78 expression showed lower and higher virulence than the parental virus, respectively, in mice, suggesting that high levels of P78 expression prevent efficient virus replication in macrophages, leading to attenuation of virus virulence. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the generation of attenuated RVFV as vaccine candidates.

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014–2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn-specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.

Global Zika virus (ZIKV) outbreaks and their strong link to microcephaly have raised major public health concerns. ZIKV has been reported to affect the innate immune responses in neural stem/progenitor cells (NS/PCs). However, it is unclear how these immune factors affect neurogenesis. In this study, we used Asian-American lineage ZIKV strain PRVABC59 to infect primary human NS/PCs originally derived from fetal brains. We found that ZIKV overactivated key molecules in the innate immune pathways to impair neurogenesis in a cell stage-dependent manner. Inhibiting the overactivated innate immune responses ameliorated ZIKV-induced neurogenesis reduction. This study thus suggests that orchestrating the host innate immune responses in NS/PCs after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology., Author summary ZIKV has been reported to affect the innate immune responses in neural stem cells. We found that inhibiting the overactivated innate immune responses ameliorated Zika virus-induced neurogenesis reduction in human neural stem cells, which are the origin of the brain. This study suggests that coordinating the host innate immune responses in neural stem cells after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings show that pi-pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes. ispartof: SCIENTIFIC REPORTS vol:11 issue:1 ispartof: location:England status: published

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

A spike protein mutation D614G became dominant in SARS-CoV-2 during the COVID-19 pandemic. However, the mutational impact on viral spread and vaccine efficacy remains to be defined. Here we engineer the D614G mutation in the SARS-CoV-2 USA-WA1/2020 strain and characterize its effect on viral replication, pathogenesis, and antibody neutralization. The D614G mutation significantly enhances SARS-CoV-2 replication on human lung epithelial cells and primary human airway tissues, through an improved infectivity of virions with the spike receptor-binding domain in an “up” conformation for binding to ACE2 receptor. Hamsters infected with D614 or G614 variants developed similar levels of weight loss. However, the G614 virus produced higher infectious titers in the nasal washes and trachea, but not lungs, than the D614 virus. The hamster results confirm clinical evidence that the D614G mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increases transmission. For antibody neutralization, sera from D614 virus-infected hamsters consistently exhibit higher neutralization titers against G614 virus than those against D614 virus, indicating that (i) the mutation may not reduce the ability of vaccines in clinical trials to protect against COVID-19 and (ii) therapeutic antibodies should be tested against the circulating G614 virus before clinical development.ImportanceUnderstanding the evolution of SARS-CoV-2 during the COVID-19 pandemic is essential for disease control and prevention. A spike protein mutation D614G emerged and became dominant soon after the pandemic started. By engineering the D614G mutation into an authentic wild-type SARS-CoV-2 strain, we demonstrate the importance of this mutation to (i) enhanced viral replication on human lung epithelial cells and primary human airway tissues, (ii) improved viral fitness in the upper airway of infected hamsters, and (iii) increased susceptibility to neutralization. Together with clinical findings, our work underscores the importance of this mutation in viral spread, vaccine efficacy, and antibody therapy.

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site (ΔPRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the ΔPRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the ΔPRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays., Article Summary: A deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

The genome of SARS-CoV-2 (SARS2) encodes for two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. NSP3 and NSP5 of SARS-CoV (SARS1) are known interferon antagonists. Here, we examined whether the protease function of SARS2 NSP3 and NSP5 target proteins involved in the host innate immune response. We designed a fluorescent based cleavage assay to rapidly screen the protease activity of NSP3 and NSP5 on a library of 71 human innate immune proteins (HIIPs), covering most pathways involved in human innate immunity. By expressing each of these HIIPs with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of NSP3 or NSP5, we could readily detect cleavage of cognate HIIPs on SDS-page gels. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type- I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of IL-6 and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in mice, the second cleavage site of NLRP12 is absent. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for in-depth studies into the pathophysiology of COVID-19 and should facilitate the search or development of more effective animal models for severe COVID-19. Finally, we discovered that one particular species of bats, David’s Myotis, possesses the five cleavage sites found in humans for NLRP12, TAB1 and IRF3. These bats are endemic from the Hubei province in China and we discuss its potential role as reservoir for the evolution of SARS1 and SASR2.

[Image: see text] We identified and explored the structure–activity-relationship (SAR) of an adamantane carboxamide chemical series of Ebola virus (EBOV) inhibitors. Selected analogs exhibited half-maximal inhibitory concentrations (EC(50) values) of ∼10–15 nM in vesicular stomatitis virus (VSV) pseudotyped EBOV (pEBOV) infectivity assays, low hundred nanomolar EC(50) activity against wild type EBOV, aqueous solubility >20 mg/mL, and attractive metabolic stability in human and nonhuman liver microsomes. X-ray cocrystallographic characterizations of a lead compound with the EBOV glycoprotein (GP) established the EBOV GP as a target for direct compound inhibitory activity and further provided relevant structural models that may assist in identifying optimized therapeutic candidates.

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways, including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host’s IFN-I response, West Nile virus (WNV) replication is sensitive to pretreatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV’s sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells, WNV replication is significantly increased and IFN-I induction and signaling are impaired compared to wild-type (wt) cells. IFN-β pretreatment was more effective in protecting against subsequent WNV infection in wt cells than TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next-generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced JAK1 and STAT1 phosphorylation and impaired induction of several interferon-stimulated genes (ISGs) following WNV infection or IFN-β treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function are dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identify VAMP8 as a novel regulator of the IFN-I system. IMPORTANCE WNV is a mosquito-borne flavivirus that poses a threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here, we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and virus-targeted antiviral strategies.

Rift Valley fever phlebovirus (RVFV) is a pathogen of Rift Valley fever, which is a mosquito-borne zoonotic disease for domestic livestock and humans in African countries. Currently, no approved vaccine is available for use in non-endemic areas. The MP-12 strain is so far the best live attenuated RVFV vaccine candidate because of its good protective efficacy in animal models. However, there are safety concerns for use of MP-12 in humans. We previously developed a single-cycle replicable MP-12 (scMP-12) which lacks NSs gene and undergoes only a single round of viral replication because of its impaired ability to induce membrane-membrane fusion. In the present study, we generated an scMP-12 mutant (scMP-12-mutNSs) carrying a mutant NSs, which degrades double-stranded RNA-dependent protein kinase R but does not inhibit host transcription. Immunization of mice with a single dose (105 PFU) of scMP-12-mutNSs elicited RVFV neutralizing antibodies and high titers of anti-N IgG production and fully protected the mice from lethal wild-type RVFV challenge. Immunogenicity and protective efficacy of scMP-12-mutNSs were better than scMP-12, demonstrating that scMP-12-mutNSs is a more efficacious vaccine candidate than scMP-12. Furthermore, our data suggested that RVFV vaccine efficacy can be improved by using this specific NSs mutant.

Background Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited. Methods Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP). Results Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection. Conclusion Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected over 200 million people throughout the world as of August 2021. There are currently no approved treatments providing high chance of recovery from a severe case of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, and the beneficial effect of Remdesivir and passive immunization therapies may only be seen when administered early on disease onset. The emergence of variants is also raising concerns regarding the efficacy of antibody therapies, antivirals, and vaccines. Therefore, there is still a need to develop new antivirals. Here, we investigated the suitability of primary human epithelial cells from the trachea/bronchia (NHBE) and small airway (SAEC) as lung models of SARS-CoV-2 infection to determine, whether the microbicide polyphenylene carboxymethylene (PPCM) has antiviral activity against SARS-CoV-2. Both NHBE and SAEC expressed proteins required for virus entry in lung epithelial cells. However, these cells were only low to moderately permissive to SARS-CoV-2 as titers increased at best by 2.5 log10 during an 8-day kinetic. Levels of replication in SAEC, unlike in NHBE, were consistent with data from other studies using human normal tissues or air-liquid interface cultures, suggesting that SAEC may be more relevant to use than NHBE for drug screening. PPCM EC50 against SARS-CoV-2 was between 32 and 132 μg/ml with a selectivity index between 12 and 41, depending on the cell type and the infective dose used. PPCM doses were consistent with those previously showing effect against other human viruses. Finally, PPCM antiviral effect observed in SAEC was in line with reduction of inflammatory markers observed overly expressed in severe COVID-19 patients. Altogether, our data support the fact that PPCM should be further evaluated in vivo for toxicity and antiviral activity against SARS-CoV-2.

We identified and explored the structure–activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus endemic to sub-Saharan Africa and the Arabian Peninsula and the etiological agent of Rift Valley fever. Rift Valley fever is a disease of major public health and economic concern, affecting livestock and humans. In ruminants, RVFV infection is characterized by high mortality rates in newborns and near 100% abortion rates in pregnant animals. Infection in humans is typically manifested as a self-limiting febrile illness, but can lead to severe and fatal hepatitis, encephalitis, hemorrhagic fever or retinitis with partial or complete blindness. Currently, there are no specific treatment options available for RVFV infection. This review presents a summary of the therapeutic approaches that have been explored on the treatment of RVFV infection.

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host’s IFN-I response, West Nile Virus (WNV) replication is sensitive to pre-treatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV’s sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells WNV replication is significantly increased and IFN-I induction and signaling is impaired compared to wild-type (wt) cells. IFNβ pre-treatment was more effective in protecting against subsequent WNV infection in wt cells as compared to TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFNβ treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function is dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system.IMPORTANCEWNV is a mosquito-borne flavivirus that poses threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and viral-targeted antiviral strategies.

Quinacrine hydrochloride is a small-molecule, orally bioavailable drug that has been used clinically as an antimalarial and for many other applications. A machine learning model trained on Ebola virus (EBOV) screening data identified quinacrine as a potent (nanomolar) in vitro inhibitor. In the current study, quinacrine 25 mg/kg was shown to protect 70% of mice (statistically significant) from a lethal challenge with mouse-adapted EBOV with once-daily intraperitoneal dosing for 8 days.

Enteric viruses exploit bacterial components including lipopolysaccharides (LPS) and peptidoglycan (PG) to facilitate infection in humans. With origins in the bat enteric system, we wondered if severe acute respiratory syndrome-coronavirus (SARS-CoV) or Middle East respiratory syndrome-CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity finding no change following LPS treatment. However, PG from B. subtilis reduced infection >10,000-fold while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity and surfactin treatment of the virus inoculum ablated infection in vivo. Finally, similar cyclic lipopeptides had no effect on CoV infectivity and the inhibitory effect of surfactin extended broadly to enveloped viruses including influenza, Ebola, Zika, Nipah, Chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad virucidal activity and suggest bacteria byproducts may negatively modulate virus infection.ImportanceIn this manuscript, we considered a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.