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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.

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.

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.

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.

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.

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.

Nipah virus (NiV) and Hendra virus are highly pathogenic zoonotic viruses of the genus Henipavirus, family Paramyxoviridae. These viruses were first identified as the causative agents of severe respiratory and encephalitic disease in the 1990s across Australia and Southern Asia with mortality rates reaching up to 75%. While outbreaks of Nipah and Hendra virus infections remain rare and sporadic, there is concern that NiV has pandemic potential. Despite increased attention, little is understood about the neuropathogenesis of henipavirus infection. Neuropathogenesis appears to arise from dual mechanisms of vascular disease and direct parenchymal brain infection, but the relative contributions remain unknown while respiratory disease arises from vasculitis and respiratory epithelial cell infection. This review will address NiV basic clinical disease, pathology and pathogenesis with a particular focus on central nervous system (CNS) infection and address the necessity of a model of relapsed CNS infection. Additionally, the innate immune responses to NiV infection in vitro and in the CNS are reviewed as it is likely linked to any persistent CNS infection.

The 2014 Ebolavirus outbreak in West Africa highlighted the need for vaccines and therapeutics to prevent and treat filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would facilitate the screening of anti-filovirus agents. To that end, we characterized knockout mice lacking &alpha, /&beta, and &gamma, interferon receptors (IFNAGR KO) as a model for wild-type filovirus infection. Intraperitoneal challenge of IFNAGR KO mice with several known human pathogenic species from the genus Ebolavirus and Marburgvirus, except Bundibugyo ebolavirus and Ta&iuml, Forest ebolavirus, caused variable mortality rate. Further characterization of the prototype Ebola virus Kikwit isolate infection in this KO mouse model showed 100% lethality down to a dilution equivalent to 1.0 &times, 10&minus, 1 pfu with all deaths occurring between 7 and 9 days post-challenge. Viral RNA was detectable in serum after challenge with 1.0 &times, 102 pfu as early as one day after infection. Changes in hematology and serum chemistry became pronounced as the disease progressed and mirrored the histological changes in the spleen and liver that were also consistent with those described for patients with Ebola virus disease. In a proof-of-principle study, treatment of Ebola virus infected IFNAGR KO mice with favipiravir resulted in 83% protection. Taken together, the data suggest that IFNAGR KO mice may be a useful model for early screening of anti-filovirus medical countermeasures.

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 absence of TRIM6 function, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite numerous mechanisms to restrict 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 important in regulating WNV replications 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. Additionally, induction of Ifnb mRNA was delayed and the expression of several IFN-stimulated genes (ISGs) was reduced in TRIM6 KO cells. IFNβ pre-treatment was more effective in protecting against subsequent WNV infection in wt cells, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next generation sequencing, we identified potential factors involved in this TRIM6-mediated antiviral response. One identified gene, VAMP8, is a soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in the vesicle-associated membrane protein subfamily. Knockdown of VAMP8 resulted in reduced STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFNβ treatment. Therefore, VAMP8 is a novel gene involved in the regulation of IFN-I signaling, and its expression is dependent on TRIM6 function. Overall, these results indicate that TRIM6 contributes to the antiviral response against WNV by regulating 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.

Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.

Summary: Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism. : Differential susceptibilities of bats to filoviruses have been suggested. Takadate et al. compare structures of the filovirus receptor among a variety of bat cell lines and discover a molecular mechanism determining their susceptibility to Ebola and Marburg viruses, providing information for understanding the ecology of filoviruses. Keywords: Ebola virus, Marburg virus, filovirus, bat, natural host, host range, glycoprotein, receptor, Niemann-Pick C1, virus-host interaction

Background La Crosse virus (LACV) causes pediatric encephalitis in the USA. LACV induces severe inflammation in the central nervous system, but the recruitment of inflammatory cells is poorly understood. A deeper understanding of LACV-induced neural pathology is needed in order to develop treatment options. However, there is a severe limitation of relevant human neuronal cell models of LACV infection. Methods We utilized human neural stem cell (hNSC)-derived neuron/astrocyte co-cultures to study LACV infection in disease-relevant primary cells. hNSCs were differentiated into neurons and astrocytes and infected with LACV. To characterize susceptibility and responses to infection, we measured viral titers and levels of viral RNA, performed immunofluorescence analysis to determine the cell types infected, performed apoptosis and cytotoxicity assays, and evaluated cellular responses to infection using qRT-PCR and Bioplex assays. Results hNSC-derived neuron/astrocyte co-cultures were susceptible to LACV infection and displayed apoptotic responses as reported in previous in vitro and in vivo studies. Neurons and astrocytes are both targets of LACV infection, with neurons becoming the predominant target later in infection possibly due to astrocytic responses to IFN. Additionally, neuron/astrocyte co-cultures responded to LACV infection with strong proinflammatory cytokine, chemokine, as well as MMP-2, MMP-7, and TIMP-1 responses. Conclusions hNSC-derived neuron/astrocyte co-cultures reproduce key aspects of LACV infection in humans and mice and are useful models to study encephalitic viruses. Specifically, we show astrocytes to be susceptible to LACV infection and that neurons and astrocytes are important drivers of the inflammatory responses seen in LACV infection through the production of proinflammatory cytokines and chemokines. Electronic supplementary material The online version of this article (10.1186/s12974-018-1356-5) contains supplementary material, which is available to authorized users.

Pulmonary fibrosis, characterized by excessive collagen deposition in the lungs, comprises a key and debilitating component of chronic lung diseases. Methods are lacking for the direct visualization of fibrillar collagen throughout the whole murine lung, a capability that would aid the understanding of lung fibrosis. We combined an optimized organ-level optical clearing (OC) approach with large-scale, label-free multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM) to reveal the complete network of fibrillar collagen in whole murine lungs. An innate inflammation-driven model based on repetitive poly(I:C) challenge was evaluated. Following OC, mosaic MPM/SHGM imaging with 3D reconstruction and whole organ quantitative analysis revealed significant differences in collagen deposition between PBS and poly(I:C) treated lungs. Airway specific analysis in whole lung acquisitions revealed significant sub-epithelial fibrosis evident throughout the proximal conductive and distal airways with higher collagen deposition in the poly(I:C) group vs PBS group. This study establishes a new, powerful approach based on OC and MPM/SHGM imaging for 3D analysis of lung fibrosis with macroscopic views of lung pathology based on microscopy and providing a new way to analyze the whole lung while avoiding regional sampling bias.

Marburg virus (MARV; family Filoviridae) causes sporadic outbreaks of Marburg hemorrhagic fever in sub-Saharan Africa with case fatality rates reaching 90%. Wild-type filoviruses, including MARV and the closely related Ebola virus, are unable to suppress the type I interferon response in rodents, and therefore require adaptation of the viruses to cause disease in immunocompetent animals. In the current study, we demonstrate that STAT2 knockout Syrian hamsters are susceptible to infection with different wild-type MARV variants. MARV Musoke causes a robust and systemic infection resulting in lethal disease. Histopathological findings share features similar to those observed in human patients and other animal models of filovirus infection. Reverse-transcription polymerase chain reaction analysis of host transcripts shows a dysregulation of the innate immune response. Our results demonstrate that the STAT2 knockout hamster represents a novel small animal model of severe MARV infection and disease without the requirement for virus adaptation.

Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machine-learning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2- and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dose-dependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was ∼2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD.

Nipah and Hendra viruses are recently emerged bat-borne paramyxoviruses (genus Henipavirus) causing severe encephalitis and respiratory disease in humans with fatality rates ranging from 40–75%. Despite the severe pathogenicity of these viruses and their pandemic potential, no therapeutics or vaccines are currently approved for use in humans. Favipiravir (T-705) is a purine analogue antiviral approved for use in Japan against emerging influenza strains; and several phase 2 and 3 clinical trials are ongoing in the United States and Europe. Favipiravir has demonstrated efficacy against a broad spectrum of RNA viruses, including members of the Paramyxoviridae, Filoviridae, Arenaviridae families, and the Bunyavirales order. We now demonstrate that favipiravir has potent antiviral activity against henipaviruses. In vitro, favipiravir inhibited Nipah and Hendra virus replication and transcription at micromolar concentrations. In the Syrian hamster model, either twice daily oral or once daily subcutaneous administration of favipiravir for 14 days fully protected animals challenged with a lethal dose of Nipah virus. This first successful treatment of henipavirus infection in vivo with a small molecule drug suggests that favipiravir should be further evaluated as an antiviral treatment option for henipavirus infections.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula that affects sheep, cattle, goats, camels, and humans. Effective vaccination of susceptible ruminants is important for the prevention of RVF outbreaks. Live-attenuated RVF vaccines are in general highly immunogenic in ruminants, whereas residual virulence might be a concern for vulnerable populations. It is also important for live-attenuated strains to encode unique genetic markers for the differentiation from wild-type RVFV strains. In this study, we aimed to strengthen the attenuation profile of the MP-12 vaccine strain via the introduction of 584 silent mutations. To minimize the impact on protective efficacy, codon usage and codon pair bias were not de-optimized. The resulting rMP12-GM50 strain showed 100% protective efficacy with a single intramuscular dose, raising a 1:853 mean titer of plaque reduction neutralization test. Moreover, outbred mice infected with one of three pathogenic reassortant ZH501 strains, which encoded rMP12-GM50 L-, M-, or S-segments, showed 90%, 50%, or 30% survival, respectively. These results indicate that attenuation of the rMP12-GM50 strain is significantly attenuated via the L-, M-, and S-segments. Recombinant RVFV vaccine strains encoding similar silent mutations will be also useful for the surveillance of reassortant strains derived from vaccine strains in endemic countries.

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.

The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors., The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses. IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors.

Hydrogen sulfide is an important endogenous mediator that has been the focus of intense investigation in the past few years, leading to the discovery of its role in vasoactive, cytoprotective and anti-inflammatory responses. Recently, we made a critical observation that H2S also has a protective role in paramyxovirus infection by modulating inflammatory responses and viral replication. In this study we tested the antiviral and anti-inflammatory activity of the H2S slow-releasing donor GYY4137 on enveloped RNA viruses from Ortho-, Filo-, Flavi- and Bunyavirus families, for which there is no FDA-approved vaccine or therapeutic available, with the exception of influenza. We found that GYY4137 significantly reduced replication of all tested viruses. In a model of influenza infection, GYY4137 treatment was associated with decreased expression of viral proteins and mRNA, suggesting inhibition of an early step of replication. The antiviral activity coincided with the decrease of viral-induced pro-inflammatory mediators and viral-induced nuclear translocation of transcription factors from Nuclear Factor (NF)-kB and Interferon Regulatory Factor families. In conclusion, increasing cellular H2S is associated with significant antiviral activity against a broad range of emerging enveloped RNA viruses, and should be further explored as potential therapeutic approach in relevant preclinical models of viral infections.

Nipah virus (NiV) is a zoonotic emerging paramyxovirus that can cause fatal respiratory illness or encephalitis in humans. Despite many efforts, the molecular mechanisms of NiV-induced acute lung injury (ALI) remain unclear. We previously showed that NiV replicates to high titers in human lung grafts in NOD-SCID/γ mice, resulting in a robust inflammatory response. Interestingly, these mice can undergo human immune system reconstitution by the bone marrow, liver, and thymus (BLT) reconstitution method, in addition to lung tissue engraftment, giving altogether a realistic model to study human respiratory viral infections. Here, we characterized NiV Bangladesh strain (NiV-B) infection of human lung grafts from human immune system-reconstituted mice in order to identify the overall effect of immune cells on NiV pathogenesis of the lung. We show that NiV-B replicated to high titers in human lung grafts and caused similar cytopathic effects irrespective of the presence of human leukocytes in mice. However, the human immune system interfered with virus spread across lung grafts, responded to infection by leukocyte migration to small airways and alveoli of the lung grafts, and accelerated oxidative stress in lung grafts. In addition, the presence of human leukocytes increased the expression of cytokines and chemokines that regulate inflammatory influx to sites of infection and tissue damage. These results advance our understanding of how the immune system limits NiV dissemination and contributes to ALI and inform efforts to identify therapeutic targets. IMPORTANCE Nipah virus (NiV) is an emerging paramyxovirus that can cause a lethal respiratory and neurological disease in humans. Only limited data are available on NiV pathogenesis in the human lung, and the relative contribution of the innate immune response and NiV to acute lung injury (ALI) is still unknown. Using human lung grafts in a human immune system-reconstituted mouse model, we showed that the NiV Bangladesh strain induced cytopathic lesions in lung grafts similar to those described in patients irrespective of the donor origin or the presence of leukocytes. However, the human immune system interfered with virus spread, responded to infection by leukocyte infiltration in the small airways and alveolar area, induced oxidative stress, and triggered the production of cytokines and chemokines that regulate inflammatory influx by leukocytes in response to infection. Understanding how leukocytes interact with NiV and cause ALI in human lung xenografts is crucial for identifying therapeutic targets.

Rift Valley fever phlebovirus (RVFV) causes high rates of abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral transmission occurs via mosquito vectors in endemic areas, which necessitates regular vaccination of susceptible livestock animals to prevent the RVF outbreaks. Although ZH501 strain has been used as a challenge strain for past vaccine efficacy studies, further characterization of other RVFV strains is important to optimize ruminant and nonhuman primate RVFV challenge models. This study aimed to characterize the virulence of wild-type RVFV strains belonging to different genetic lineages in outbred CD1 mice. Mice were intraperitoneally infected with 1x103 PFU of wild-type ZH501, Kenya 9800523, Kenya 90058, Saudi Arabia 200010911, OS1, OS7, SA75, Entebbe, or SA51 strains. Among them, mice infected with SA51, Entebbe, or OS7 strain showed rapid dissemination of virus in livers and peracute necrotic hepatitis at 2–3 dpi. Recombinant SA51 (rSA51) and Zinga (rZinga) strains were recovered by reverse genetics, and their virulence was also tested in CD1 mice. The rSA51 strain reproduced peracute RVF disease in mice, whereas the rZinga strain showed a similar virulence with that of rZH501 strain. This study showed that RVFV strains in different genetic lineages display distinct virulence in outbred mice. Importantly, since wild-type RVFV strains contain defective-interfering RNA or various genetic subpopulations during passage from original viral isolations, recombinant RVFV strains generated by reverse genetics will be better suitable for reproducible challenge studies for vaccine development as well as pathological studies.

Ebola virus disease (EVD) is caused by Ebola virus (EBOV) and characterized in humans by hemorrhagic fever with high fatality rates. Human-to-human EBOV transmission occurs by physical contact with infected body fluids, or indirectly by contaminated surfaces. Sexual transmission is a route of infection only recently documented despite isolating EBOV virus or genome in the semen since 1976. Data on dissemination of EBOV from survivors remain limited and EBOV pathogenesis in humans following sexual transmission is unknown. The in vitro antiviral efficacy of polyphenylene carboxymethylene (PPCM) against EBOV was investigated considering the limited countermeasures available to block infection through sexual intercourse. PPCM is a vaginal topical contraceptive microbicide shown to prevent sexual transmission of HIV, herpes virus, and bacterial infections in several different models. Here we demonstrate its antiviral activity against EBOV. No viral replication was detected in the presence of PPCM in cell culture, including vaginal epithelial (VK2/E6E7) cells. Specifically, PPCM reduced viral attachment to cells by interfering with EBOV glycoprotein, and possibly through binding the cell surface glycosaminoglycan heparan sulfate important in the infection process. EBOV-infected VK2/E6E7 cells were found to secrete type III interferon (IFN), suggesting activation of distinct PRRs or downstream signaling factors from those required for type I and II IFN. The addition of PPCM following cell infection prevented notably the increase of these inflammation markers. Therefore, PPCM could potentially be used as a topical microbicide to reduce transmission by EBOV-positive survivors during sexual intercourse.

Interferons (IFN) are essential antiviral cytokines that establish the cellular antiviral state through upregulation of hundreds of interferon-stimulated genes (ISGs), most of which have uncharacterized functions and mechanisms. We identified Cholesterol-25-hydroxylase (Ch25h) as an antiviral ISG that can convert cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). Ch25h expression or 25HC treatment in cultured cells broadly inhibits enveloped viruses including VSV, HSV, HIV, and MHV68 as well as acutely pathogenic EBOV, RVFV, RSSEV, and Nipah viruses under BSL4 conditions. As a soluble oxysterol, 25HC inhibits viral entry by blocking membrane fusion between virus and cell. In animal models, Ch25h-knockout mice were more susceptible to MHV68 lytic infection. Moreover, administration of 25HC in humanized mice suppressed HIV replication and rescued T-cell depletion. Thus, our studies demonstrate a unique mechanism by which IFN achieves its antiviral state through the production of a natural oxysterol to inhibit viral entry and implicate membrane-modifying oxysterols as potential antiviral therapeutics.

The tick-borne encephalitis complex contains a number of flaviviruses that share close genetic homology, and are responsible for significant human morbidity and mortality with widespread geographical range. Although many members of this complex have been recognised for decades, licenced human vaccines with broad availability are only available for tick-borne encephalitis virus. While tick-borne encephalitis virus vaccines have been demonstrated to induce significant protective immunity, as determined by virus-neutralisation titres, vaccine breakthrough (clinical infection following complete vaccination), has been described. The aim of this study was to confirm the cross-neutralisation of tick-borne flaviviruses using mouse immune ascitic fluids, and to determine the magnitude of cross-neutralising antibody titres in sera from donors following tick-borne encephalitis vaccination, infection, and vaccine breakthrough. The results demonstrate that there is significant cross-neutralisation of representative members of the tick-borne encephalitis complex following vaccination and/or infection, and that the magnitude of immune responses varies based upon the exposure type. Donor sera successfully neutralised most of the viruses tested, with 85% of vaccinees neutralising Kyasanur forest disease virus and 73% of vaccinees neutralising Alkhumra virus. By contrast, only 63% of vaccinees neutralised Powassan virus, with none of these neutralisation titres exceeding 1:60. Taken together, the data suggest that tick-borne encephalitis virus vaccination may protect against most of the members of the tick-borne encephalitis complex including Kyasanur forest disease virus and Alkhumra virus, but that the neutralisation of Powassan virus following tick-borne encephalitis vaccination is minimal., Tick-borne encephalitis: One vaccine protects against multiple flaviviruses Prior exposure to tick-borne encephalitis virus confers immunity to distinct, but genetically related, significant human pathogens. The tick-borne encephalitis complex comprises multiple ‘flaviviruses’; however, a licensed vaccine only exists for one: tick-borne encephalitis virus (TBEV). Alexander McAuley, of the University of Texas Medical Branch, USA, and collaborators tested human serum samples from donors who had either received the TBEV vaccine, been infected with the virus naturally, or had contracted the virus despite prior vaccination. The team tested the sera against six genetically related flaviviruses, looking for antibodies that indicate viral immunity. The results showed that TBEV exposure induce production of antibodies that inhibit other flaviviruses, the efficacy of which decreases as the genetic variation from TBEV increases. Additionally, sera from infected individuals generated more antibodies than sera from vaccinated donors. This study provides an insight into the relationship between genetic similarity and vaccine cross-protection.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections., Author Summary Nipah virus (NiV) is a zoonotic paramyxovirus causing severe respiratory and encephalitic illness with case fatality rates of 40 to 90%. The host type-I interferon (IFN-I) system protects against viral infections; however, to establish productive infection NiV has developed mechanisms to evade these host antiviral responses. An important component of the IFN system is the IKKε kinase, which is directly involved in IFN-I production and IFN-I signaling. The activity of the IKKε kinase is regulated by unanchored K48-linked polyubiquitin chains, a novel form of ubiquitin that is not covalently attached to any protein and can induce activation of kinases by promoting protein oligomerization. These unanchored polyubiquitin chains that activate IKKε are generated by the E3-ubiquitin ligase TRIM6. Here we demonstrate that the matrix structural protein (M) of NiV, which is important for virus assembly and budding, also has IFN-I antagonist functions and interferes with the host antiviral response. We found that NiV-M interacts with TRIM6 and promotes its degradation. Consequently, association of unanchored polyubiquitin chains with IKKε is reduced leading to impaired IKKε activation and ineffective IFN responses. Since the matrix protein is present in the virions and is released immediately after virus entry into the cell, this provides an efficient mechanism to escape the host antiviral response. These data may help explain the highly pathogenic potential of these viruses.

ISG15 is an interferon (IFN)-α/β-induced ubiquitin-like protein. It exists as a free molecule, intracellularly and extracellularly, and conjugated to target proteins. Studies in mice have demonstrated a role for Isg15 in antiviral immunity. By contrast, human ISG15 was shown to have critical immune functions, but not in antiviral immunity. Namely, free extracellular ISG15 is crucial in IFN-γ-dependent antimycobacterial immunity, while free intracellular ISG15 is crucial for USP18-mediated downregulation of IFN-α/β signalling. Here we describe ISG15-deficient patients who display no enhanced susceptibility to viruses in vivo, in stark contrast to Isg15-deficient mice. Furthermore, fibroblasts derived from ISG15-deficient patients display enhanced antiviral protection, and expression of ISG15 attenuates viral resistance to WT control levels. The species-specific gain-of-function in antiviral immunity observed in ISG15 deficiency is explained by the requirement of ISG15 to sustain USP18 levels in humans, a mechanism not operating in mice., ISG15 is a ubiquitin-like protein which has important immune-related functions in mice and humans. Here the authors demonstrate that, unlike in mice, human ISG15 stabilizes UPS18 and that ISG15-deficient human cells are more resistant to viral infection.

The budding of Nipah virus, a deadly member of the Henipavirus genus within the Paramyxoviridae, has been thought to be independent of the host ESCRT pathway, which is critical for the budding of many enveloped viruses. This conclusion was based on the budding properties of the virus matrix protein in the absence of other virus components. Here, we find that the virus C protein, which was previously investigated for its role in antagonism of innate immunity, recruits the ESCRT pathway to promote efficient virus release. Inhibition of ESCRT or depletion of the ESCRT factor Tsg101 abrogates the C enhancement of matrix budding and impairs live Nipah virus release. Further, despite the low sequence homology of the C proteins of known henipaviruses, they all enhance the budding of their cognate matrix proteins, suggesting a conserved and previously unknown function for the henipavirus C proteins., Author Summary Nipah virus is a deadly pathogen (40–100% mortality) that has yearly outbreaks in Southeast Asia, resulting from spillover from its natural fruit bat reservoir. The viral C protein is one of only nine virus proteins, but its role in promoting virus replication is not fully understood. Here, we found that the C protein promotes the efficient release of budding Nipah virus from infected cells. It does so by recruiting an essential factor in the host ESCRT complex, Tsg101. The ESCRT complex has well-characterized functions in mediating membrane pinching off events that resemble virus budding. Further, we found that the C proteins of related viruses within the same genus (Henipavirus) also promote virus budding, suggesting that this previously unknown function of the henipavirus C proteins is conserved. This work illuminates the basic biology of henipaviruses with significant outbreak and public health concern, and opens the door to further lines of inquiry.

Incorporation of reporter genes within virus genomes is an indispensable tool for interrogation of virus biology and pathogenesis. In previous work, we incorporated a fluorophore into a viral ORF by attaching it to the viral gene via a P2A ribosomal skipping sequence. This recombinant Nipah virus, however, was attenuated in vitro relative to WT virus. In this work, we determined that inefficient ribosomal skipping was a major contributing factor to this attenuation. Inserting a GSG linker before the P2A sequence resulted in essentially complete skipping, significantly improved growth in vitro, and WT lethality in vivo. To the best of our knowledge, this represents the first time a recombinant virus of Mononegavirales with integration of a reporter into a viral ORF has been compared with the WT virus in vivo. Incorporating the GSG linker for improved skipping efficiency whenever functionally important is a critical consideration for recombinant virus design.

The recent epidemic of Ebola has intensified the need for the development of novel antiviral therapeutics that prolong and improve survival against deadly viral diseases. We sought to determine whether ranpirnase, an endoribonuclease from Rana pipiens with a demonstrated human safety profile in phase III oncology trials, can reduce titers of Ebola virus (EBOV) in infected cells, protect mice against mouse-adapted EBOV challenge, and reduce virus levels in infected mice. Our results demonstrate that 0.50 μg/ml ranpirnase is potently effective at reducing EBOV Zaire Kikwit infection in cultured Vero E6 cells (Selectivity Index 47.8–70.2). In a prophylactic study, a single intravenous dose of 0.1 mg/kg ranpirnase protected 70% of mice from progressive infection. Additionally, in a post-exposure prophylactic study, 100% of female mice survived infection after intraperitoneal administration of 0.1 mg/kg ranpirnase for ten days beginning 1 h post challenge. Most of the male counterparts were sacrificed due to weight loss by Study Day 8 or 9; however, the Clinical Activity/Behavior scores of these mice remained low and no significant microscopic pathologies could be detected in the kidneys, livers or spleens. Furthermore, live virus could not be detected in the sera of ranpirnase-treated mice by Study Day 8 or in the kidneys, livers or spleens by Study Day 12, and viral RNA levels declined exponentially by Study Day 12. Because ranpirnase is exceptionally stable and has a long track record of safe intravenous administration to humans, this drug provides a promising new candidate for clinical consideration in the treatment of Ebola virus disease alone or in combination with other therapeutics.

Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human veterinary pathogen causing acute hepatitis in ruminants and has the potential to Single-particle cryo-EM reconstruction of RVFV MP-12 hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T=12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit-vaccines.

Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans, and a high rate of abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is attenuated in the M- and L-segments, but the S-segment retains the virulent phenotype. MP-12 was manufactured as an Investigational New Drug vaccine by using MRC-5 cells and encodes a functional NSs gene, the major virulence factor of RVFV which 1) induces a shutoff of the host transcription, 2) inhibits interferon (IFN)-β promoter activation, and 3) promotes the degradation of dsRNA-dependent protein kinase (PKR). MP-12 lacks a marker for differentiation of infected from vaccinated animals (DIVA). Although MP-12 lacking NSs works for DIVA, it does not replicate efficiently in type-I IFN-competent MRC-5 cells, while the use of type-I IFN-incompetent cells may negatively affect its genetic stability. To generate modified MP-12 vaccine candidates encoding a DIVA marker, while still replicating efficiently in MRC-5 cells, we generated recombinant MP-12 encoding Punta Toro virus Adames strain NSs (rMP12-PTNSs) or Sandfly fever Sicilian virus NSs (rMP12-SFSNSs) in place of MP-12 NSs. We have demonstrated that those recombinant MP-12 viruses inhibit IFN-β mRNA synthesis, yet do not promote the degradation of PKR. The rMP12-PTNSs, but not rMP12-SFSNSs, replicated more efficiently than recombinant MP-12 lacking NSs in MRC-5 cells. Mice vaccinated with rMP12-PTNSs or rMP12-SFSNSs induced neutralizing antibodies at a level equivalent to those vaccinated with MP-12, and were efficiently protected from wild-type RVFV challenge. The rMP12-PTNSs and rMP12-SFSNSs did not induce antibodies cross-reactive to anti-RVFV NSs antibody and are therefore applicable to DIVA. Thus, rMP12-PTNSs is highly efficacious, replicates efficiently in MRC-5 cells, and encodes a DIVA marker, all of which are important for vaccine development for Rift Valley fever., Author Summary Upon outbreak of zoonotic viral diseases in herds of animals, early detection of naturally infected animals and prevention of further viral spread are important for minimizing the impact of outbreak in the society. Vaccination may compromise the identification of infected animals since both natural infection and vaccination induce antibodies specific to the pathogen. Therefore, new generation vaccines should have a marker to differentiate infected from vaccinated animals (DIVA). Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen which can cause hemorrhagic fever, neurological disorders or blindness in humans and a high-rate abortion in ruminants. MP-12 strain, a live-attenuated candidate vaccine, is safe and immunogenic, but lacks a DIVA marker. In this study, we developed and characterized improved MP-12 viruses which encode a DIVA marker by replacing the virulence gene with that of serologically distinct viruses belonging to the same genera. The novel MP-12 variant with such DIVA marker was highly efficacious and replicated efficiently in human diploid cells for vaccine production, and will become alternative candidate vaccines of MP-12 for veterinary applications.

© 2013 Vigant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50≤0.5 mM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001’s specific inhibition of viruscell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen (1O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated 1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. 1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced 1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001’s mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001’s limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50, This work was supported by NIH grants U01 AI070495, U01 AI082100, R01 AI069317, U54 AI065359 (PSWRCE) (to BL), AI075647 (to MK), NIH-NIGMS 5SC1GM084776 (to DZ and MS), and by Fundação para a Ciência e a Tecnologia – Ministério da Educação e Ciêcia (Portugal) project PTDC/SAU-BEB/099142/2008 (to NCS) and fellowship SFRH/BPD/72037/2010 (to AH) and Veterans Affairs research funds (SC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)–any amino acid (X)–serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

The aim of this study was to evaluate the contribution of positively charged amino acid residues for the Uukuniemi virus (UUKV) N protein functionality. Based on phlebovirus nucleocapsid (N) protein alignments and 3D-structure predictions of UUKV N protein, 14 positively charged residues were chosen as targets for the mutagenesis. The impact of mutations to the N protein functionality was analyzed using minigenome-, virus-like particle-, and mammalian two-hybrid-assays. Seven of the mutations affected the functional competence in all three assays, while others had milder impact or no impact at all. In the 3D-model of UUKV N protein, five of the affected residues, R61, R64, R73, R98 and R115, were located either within or in close proximity to the central cavity that could potentially bind RNA.

Rift Valley fever virus (RVFV) is a bunyavirus endemic to Africa and the Arabian Peninsula that infects humans and livestock. The virus encodes two glycoproteins, Gn and Gc, which represent the major structural antigens and are responsible for host cell receptor binding and fusion. Both glycoproteins are organized on the virus surface as cylindrical hollow spikes that cluster into distinct capsomers with the overall assembly exhibiting an icosahedral symmetry. Currently, no experimental three-dimensional structure for any entire bunyavirus glycoprotein is available. Using fold recognition, we generated molecular models for both RVFV glycoproteins and found significant structural matches between the RVFV Gn protein and the influenza virus hemagglutinin protein and a separate match between RVFV Gc protein and Sindbis virus envelope protein E1. Using these models, the potential interaction and arrangement of both glycoproteins in the RVFV particle was analyzed, by modeling their placement within the cryo-electron microscopy density map of RVFV. We identified four possible arrangements of the glycoproteins in the virion envelope. Each assembly model proposes that the ectodomain of Gn forms the majority of the protruding capsomer and that Gc is involved in formation of the capsomer base. Furthermore, Gc is suggested to facilitate intercapsomer connections. The proposed arrangement of the two glycoproteins on the RVFV surface is similar to that described for the alphavirus E1-E2 proteins. Our models will provide guidance to better understand the assembly process of phleboviruses and such structural studies can also contribute to the design of targeted antivirals.

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model., Author Summary Rift Valley fever virus (RVFV) is a deadly virus that is found primarily in sub-Saharan Africa. However, mosquitoes that are capable of transmitting RVFV have a worldwide distribution. RVF affects a broad range of animal species and is known to cause encephalitis or hemorrhagic fever in humans with survivors having long-term health effects such as a loss of vision. There is no vaccine currently approved for use in humans. In this study we evaluated the host response to infection with either a RVFV vaccine strain or a wild-type strain to identify similarities or differences that could be exploited for development of therapeutics or improved vaccines. While mice infected with the vaccine strain did not develop disease and survived, infection with the wild-type strain caused severe disease with a fatal outcome. Analyzing multiple clinical factors and the host immune response over the course of infection allowed us to identify potential host factors associated with disease progression during wild-type virus infection. This work also provides support for a current vaccine candidate, MP-12, in demonstrating a limited and protective host response to infection.

Background Uukuniemi virus (UUKV) belongs to the Phlebovirus genus in the family Bunyaviridae. As a non-pathogenic virus for humans UUKV has served as a safe model bunyavirus in a number of studies addressing fundamental questions such as organization and regulation of viral genes, genome replication, structure and assembly. The present study is focused on the oligomerization of the UUKV nucleocapsid (N) protein, which plays an important role in several steps of virus replication. The aim was to locate the domains involved in the N protein oligomerization and study the process in detail. Results A set of experiments concentrating on the N- and C-termini of the protein was performed, first by completely or partially deleting putative N-N-interaction domains and then by introducing point mutations of amino acid residues. Mutagenesis strategy was based on the computer modeling of secondary and tertiary structure of the N protein. The N protein mutants were studied in chemical cross-linking, immunofluorescence, mammalian two-hybrid, minigenome, and virus-like particle-forming assays. The data showed that the oligomerization ability of UUKV-N protein depends on the presence of intact α-helices on both termini of the N protein molecule and that a specific structure in the N-terminal region plays a crucial role in the N-N interaction(s). This structure is formed by two α-helices, rich in amino acid residues with aromatic (W7, F10, W19, F27, F31) or long aliphatic (I14, I24) side chains. Furthermore, some of the N-terminal mutations (e.g. I14A, I24A, F31A) affected the N protein functionality both in mammalian two-hybrid and minigenome assays. Conclusions UUKV-N protein has ability to form oligomers in chemical cross-linking and mammalian two-hybrid assays. In mutational analysis, some of the introduced single-point mutations abolished the N protein functionality both in mammalian two-hybrid and minigenome assays, suggesting that especially the N-terminal region of the UUKV-N protein is essential for the N-N interaction.

Nipah virus (NiV) and Hendra virus (HeV) are the only paramyxoviruses requiring Biosafety Level 4 (BSL-4) containment. Thus, study of henipavirus entry at less than BSL-4 conditions necessitates the use of cell-cell fusion or pseudotyped reporter virus assays. Yet, these surrogate assays may not fully emulate the biological properties unique to the virus being studied. Thus, we developed a henipaviral entry assay based on a β-lactamase-Nipah Matrix (βla-M) fusion protein. We first codon-optimized the bacterial βla and the NiV-M genes to ensure efficient expression in mammalian cells. The βla-M construct was able to bud and form virus-like particles (VLPs) that morphologically resembled paramyxoviruses. βla-M efficiently incorporated both NiV and HeV fusion and attachment glycoproteins. Entry of these VLPs was detected by cytosolic delivery of βla-M, resulting in enzymatic and fluorescent conversion of the pre-loaded CCF2-AM substrate. Soluble henipavirus receptors (ephrinB2) or antibodies against the F and/or G proteins blocked VLP entry. Additionally, a Y105W mutation engineered into the catalytic site of βla increased the sensitivity of our βla-M based infection assays by 2-fold. In toto, these methods will provide a more biologically relevant assay for studying henipavirus entry at less than BSL-4 conditions.

The phosphoinositide-3 kinase (PI3K) pathway regulates diverse cellular activities related to cell growth, migration, survival, and vesicular trafficking. It is known that Ebola virus requires endocytosis to establish an infection. However, the cellular signals that mediate this uptake were unknown for Ebola virus as well as many other viruses. Here, the involvement of PI3K in Ebola virus entry was studied. A novel and critical role of the PI3K signaling pathway was demonstrated in cell entry of Zaire Ebola virus (ZEBOV). Inhibitors of PI3K and Akt significantly reduced infection by ZEBOV at an early step during the replication cycle. Furthermore, phosphorylation of Akt-1 was induced shortly after exposure of cells to radiation-inactivated ZEBOV, indicating that the virus actively induces the PI3K pathway and that replication was not required for this induction. Subsequent use of pseudotyped Ebola virus and/or Ebola virus-like particles, in a novel virus entry assay, provided evidence that activity of PI3K/Akt is required at the virus entry step. Class 1A PI3Ks appear to play a predominant role in regulating ZEBOV entry, and Rac1 is a key downstream effector in this regulatory cascade. Confocal imaging of fluorescently labeled ZEBOV indicated that inhibition of PI3K, Akt, or Rac1 disrupted normal uptake of virus particles into cells and resulted in aberrant accumulation of virus into a cytosolic compartment that was non-permissive for membrane fusion. We conclude that PI3K-mediated signaling plays an important role in regulating vesicular trafficking of ZEBOV necessary for cell entry. Disruption of this signaling leads to inappropriate trafficking within the cell and a block in steps leading to membrane fusion. These findings extend our current understanding of Ebola virus entry mechanism and may help in devising useful new strategies for treatment of Ebola virus infection., Author Summary Each year, filoviruses such as Ebola virus claim many human lives and decimate gorilla populations in Africa. Infection results in an acute fever often associated with profuse internal and external bleeding and death rates of up to 90%. Due to these symptoms and high pathogenicity, these viruses have been heavily publicized in the media. The first step of infection is entry, where the virus is taken up and penetrates into the cell, from which it spreads throughout the body. While it is known that the cell must engulf the virus by the process of endocytosis, we know little about how the virus triggers this event. Here, we use a novel technology to measure penetration of Ebola virus into the cell in real time and show that Ebola virus stimulates phosphoinositide-3 kinase, a signaling molecule known to induce endocytosis. Importantly, drugs that interfere with this signaling inhibit infection by Ebola virus and block virus spread. This work provides a mechanistic insight into how Ebola virus manipulates the cell to start an infection, may explain part of virus induced pathogenesis, and provides a potential way to treat this deadly disease.

Background Junín virus (JUNV), the etiologic agent of Argentine hemorrhagic fever (AHF), is classified by the NIAID and CDC as a Category A priority pathogen. Presently, antiviral therapy for AHF is limited to immune plasma, which is readily available only in the endemic regions of Argentina. T-705 (favipiravir) is a broadly active small molecule RNA-dependent RNA polymerase inhibitor presently in clinical evaluation for the treatment of influenza. We have previously reported on the in vitro activity of favipiravir against several strains of JUNV and other pathogenic New World arenaviruses. Methodology/Principal Findings To evaluate the efficacy of favipiravir in vivo, guinea pigs were challenged with the pathogenic Romero strain of JUNV, and then treated twice daily for two weeks with oral or intraperitoneal (i.p.) favipiravir (300 mg/kg/day) starting 1–2 days post-infection. Although only 20% of animals treated orally with favipiravir survived the lethal challenge dose, those that succumbed survived considerably longer than guinea pigs treated with placebo. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by i.p. injection, i.p. treatment resulted in a substantially higher level of protection (78% survival). Survival in guinea pigs treated with ribavirin was in the range of 33–40%. Favipiravir treatment resulted in undetectable levels of serum and tissue viral titers and prevented the prominent thrombocytopenia and leucopenia observed in placebo-treated animals during the acute phase of infection. Conclusions/Significance The remarkable protection afforded by i.p. favipiravir intervention beginning 2 days after challenge is the highest ever reported for a small molecule antiviral in the difficult to treat guinea pig JUNV challenge model. These findings support the continued development of favipiravir as a promising antiviral against JUNV and other related arenaviruses., Author Summary Argentine hemorrhagic fever (AHF) is a severe and often-fatal disease caused by infection with Junín virus (JUNV). Presently, there is an unmet need to develop new therapeutics to address current medical, public health and national security concerns, as JUNV is considered a potential bioterror agent amenable to aerosolization and intentional release. In the present study, favirpiravir, a promising anti-JUNV drug in clinical development for the treatment of influenza, was evaluated in an experimental small animal model of AHF. Guinea pigs challenged with JUNV were treated with favipiravir twice daily for two weeks starting 1–2 days after infection. Consistent with pharmacokinetic analysis that showed greater plasma levels of favipiravir in animals dosed by intraperitoneal injection, administration by this route resulted in a dramatic protective effect as 78% animals survived the infection compared to 11% in the placebo-treated group. Favipiravir treatment inhibited JUNV replication and prevented the development of disease observed in animals receiving placebo during the acute stage of infection. The high level efficacy observed following post-exposure prophylaxis with favipiravir is the highest ever reported for a small molecule antiviral in the guinea pig JUNV challenge model and thus supports its continued development as a promising antiviral therapy for the treatment of AHF.

Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an “off-the-shelf” therapeutic against acute NiV infection., Author Summary Nipah virus (NiV) is a lethal, newly emerging virus that causes fatal inflammation of the brain and has a high death rate in infected humans. NiV and the closely related Hendra virus (HeV) can also infect agriculturally important livestock such as pigs and horses. The lack of effective vaccines and treatments, and the ongoing threat they pose to both agriculture and public health, have led to the classification of NiV and HeV as Biosafety Level 4 (BSL4) pathogens. Paramyxoviruses such as NiV are known to replicate in the cytoplasm and bud from the plasma membrane. Viral assembly and budding is mediated by the matrix structural protein. However, we found, quite unexpectedly, that the matrix protein of NiV needs to transit through the nucleus before gaining the functional ability to localize and bud from the plasma membrane. Although NiV-M has putative nuclear import and export signals, we also found that ubiquitination of a conserved lysine residue in NiV-M is critical for nuclear export, subsequent membrane localization and viral budding. Proteasome inhibitors, which deplete cellular pools of free ubiquitin, potently reduce viral titers during live NiV infection, opening up new possibilities for therapeutics against acute NiV infection.

In this study we report the development and optimization of two minigenome rescue systems for Nipah virus, a member of the Paramyxoviridae family. One is mediated by the T7 RNA polymerase supplied either by a constitutively expressing cell line or by transfection of expression plasmids and is thus independent from infection with a helper virus. The other approach is based on RNA polymerase I-driven transcription, a unique approach for paramyxovirus reverse genetics technology. Minigenome rescue was evaluated by reporter gene activities of (i) the two different minigenome transcription systems, (ii) genomic versus antigenomic-oriented minigenomes, (iii) different ratios of the viral protein expression plasmids, and (iv) time course experiments. The high efficiency and reliability of the established systems allowed for downscaling to 96-well plates. This served as a basis for the development of a high-throughput screening system for potential antivirals that target replication and transcription of Nipah virus without the need of high bio-containment. Using this system we were able to identify two compounds that reduced minigenome activity.

Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins GN and GC, nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.