Search

RNA viruses present a constant threat to human health, often with limited options for vaccination or therapy. Notable examples include influenza viruses and coronaviruses, which have pandemic potential. Filo- and henipaviruses cause more limited outbreaks, but with high case fatality rates. All RNA viruses rely on the activity of a virus-encoded RNA-dependent RNA polymerase (RdRp). An antiviral nucleoside analogue, 4'-Fluorouridine (4'-FlU), targets RdRp and diminishes the replication of several RNA viruses, including influenza A virus and SARS-CoV-2, through incorporation into nascent viral RNA and delayed chain termination. However, the effective concentration of 4'-FlU varied among different viruses, raising the need to fortify its efficacy. Here we show that inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme essential for pyrimidine biosynthesis, can synergistically enhance the antiviral effect of 4'-FlU against influenza A viruses, SARS-CoV-2, henipaviruses, and Ebola virus. Even 4'-FlU-resistant mutant influenza A virus was re-sensitized towards 4'-FlU by DHODH inhibition. The addition of uridine rescued influenza A virus replication, strongly suggesting uridine depletion as a mechanism of this synergy. 4'-FlU was also highly effective against SARS-CoV-2 in a hamster model of COVID. We propose that the impairment of endogenous uridine synthesis by DHODH inhibition enhances the incorporation of 4'-FlU into viral RNAs. This strategy may be broadly applicable to enhance the efficacy of pyrimidine nucleoside analogues for antiviral therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: L.S., A.D., H.L.F. and M.D. are employees of University Medical Center Göttingen, which has filed a patent application covering the combination of inhibitors of pyrimidine synthesis and nucleoside analogues to treat viral infections (inventors: MD, AD). The other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Here, we describe the transcription- and replication-competent virus-like particle (trVLP) system for Marburg virus (MARV), which recapitulates transcription and replication of the viral genome in addition to viral particle assembly, egress, and entry. This protocol includes instructions for transfections for producer and acceptor cells and the use of trVLPs for infection., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Respiratory syncytial virus (RSV) selectively targets ciliated cells in human bronchial epithelium and can cause bronchiolitis and pneumonia, mostly in infants. To identify molecular targets of intervention during RSV infection in infants, we investigated how age regulates RSV interaction with the bronchial epithelium barrier. Employing precision-cut lung slices and air-liquid interface cultures generated from infant and adult human donors, we found robust RSV virus spread and extensive apoptotic cell death only in infant bronchial epithelium. In contrast, adult bronchial epithelium showed no barrier damage and limited RSV infection. Single nuclear RNA-Seq revealed age-related insufficiency of an antiapoptotic STAT3 activation response to RSV infection in infant ciliated cells, which was exploited to facilitate virus spread via the extruded apoptotic ciliated cells carrying RSV. Activation of STAT3 and blockade of apoptosis rendered protection against severe RSV infection in infant bronchial epithelium. Lastly, apoptotic inhibitor treatment of a neonatal mouse model of RSV infection mitigated infection and inflammation in the lung. Taken together, our findings identify a STAT3-mediated antiapoptosis pathway as a target to battle severe RSV disease in infants.

Filoviruses are some of the most lethal viruses in the modern world, and increasing numbers of filovirus species and genera have been discovered in recent years. Despite the potential severity of filovirus outbreaks in the human population, comparably few sensitive pan-filovirus RT-PCR assays have been described that might facilitate early detection and prevention. Here, we present a new pan-filovirus RT-PCR assay targeting the L polymerase gene for detection of all known mammalian filoviruses. We demonstrate the detection of 10 synthetic filovirus RNA templates with analytical sensitivity ranging from 178 to 3,354 copies/mL, without cross-reactivity on 10 non-filoviral human viral species. We verified assay performance on 10 inactivated filovirus isolates, yielding initial sensitivities of 0.012-44.17 TCID 50 /mL. We coupled this broadly reactive RT-PCR with a deep sequencing workflow that is amenable to high-throughput pooling to maximize detection and discovery potential. In summary, this pan-filovirus RT-PCR assay targets the most conserved filovirus gene, offers the widest breadth of coverage to date, and may help in the detection and discovery of novel filoviruses.IMPORTANCEFiloviruses remain some of the most mysterious viruses known to the world, with extremely high lethality rates and significant pandemic potential. Yet comparably few filovirus species and genera have been discovered to date and questions surround the definitive host species for zoonotic infections. Here, we describe a novel broadly reactive RT-PCR assay targeting the conserved L polymerase gene for high-throughput screening for filoviruses in a variety of clinical and environmental specimens. We demonstrate the assay can detect all known mammalian filoviruses and determine the sensitivity and specificity of the assay on synthetic RNA sequences, inactivated filovirus isolates, and non-filoviral species., Competing Interests: The authors declare no conflict of interest.

Recent advances in high-throughput sequencing technologies have led to the discovery of a plethora of previously unknown viruses in animal samples. Some of these newly detected viruses are closely related to human pathogens. A prime example are the henipaviruses. Both Nipah (NiV) and Hendra virus (HeV) cause severe disease in humans. Henipaviruses are of zoonotic origin, and animal hosts, including intermediate hosts, play a critical role in viral transmission to humans. The natural reservoir hosts of NiV and HeV seem to be restricted to a few fruit bat species of the Pteropus genus in distinct geographic areas. However, the recent discovery of novel henipa- and henipa-like viruses suggests that these viruses are far more widespread than was originally thought. To date, these new viruses have been found in a wide range of animal hosts, including bats, shrews, and rodents in Asia, Africa, Europe, and South America. Since these viruses are closely related to human pathogens, it is important to learn whether they pose a threat to human health. In this article, we summarize what is known about the newly discovered henipaviruses, highlight differences to NiV and HeV, and discuss their pathogenic potential.

Although next-generation sequencing (NGS) has been instrumental in determining the genomic sequences of emerging RNA viruses, de novo sequence determination often lacks sufficient coverage of the 5' and 3' ends of the viral genomes. Since the genome ends of RNA viruses contain the transcription and genome replication promoters that are essential for viral propagation, a lack of terminal sequence information hinders the efforts to study the replication and transcription mechanisms of emerging and re-emerging viruses. To circumvent this, we have developed a novel method termed ViBE-Seq (Viral Bona Fide End Sequencing) for the high-resolution sequencing of filoviral genome ends using a simple yet robust protocol with high fidelity. This technique allows for sequence determination of the 5' end of viral RNA genomes and mRNAs with as little as 50 ng of total RNA. Using the Ebola virus and Marburg virus as prototypes for highly pathogenic, re-emerging viruses, we show that ViBE-Seq is a reliable technique for rapid and accurate 5' end sequencing of filovirus RNA sourced from virions, infected cells, and tissue obtained from infected animals. We also show that ViBE-Seq can be used to determine whether distinct reverse transcriptases have terminal deoxynucleotidyl transferase activity. Overall, ViBE-Seq will facilitate the access to complete sequences of emerging viruses.

In tervening pro teins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCE In tervening pro teins (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity., Competing Interests: The authors declare no conflict of interest.

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-I and IFN-III responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14's antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1) and several negative-sense RNA viruses, including vesicular stomatitis virus (VSV), Ebola virus (EBOV), and Nipah virus (NiV), to infect A549 PARP14 knockout (KO) cells. HSV-1 had increased replication in PARP14 KO cells, indicating that PARP14 restricts HSV-1 replication. In contrast, PARP14 was critical for the efficient infection of VSV, EBOV, and NiV, with EBOV infectivity at less than 1% of WT cells. A PARP14 active site inhibitor had no impact on HSV-1 or EBOV infection, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both pro- and anti-viral functions targeting multiple viruses., Competing Interests: Competing interests: ARF is in the process of submitting a patent application for use of SARS-CoV-2 ΔMac1 virus as a live-attenuated vaccine.

Filoviridae is a family of negative-sense RNA viruses with genomes of about 13.1-20.9 kb that infect fish, mammals and reptiles. The filovirid genome is a linear, non-segmented RNA with five canonical open reading frames (ORFs) that encode a nucleoprotein (NP), a polymerase cofactor (VP35), a glycoprotein (GP 1,2 ), a transcriptional activator (VP30) and a large protein (L) containing an RNA-directed RNA polymerase (RdRP) domain. All filovirid genomes encode additional proteins that vary among genera. Several filovirids (e.g., Ebola virus, Marburg virus) are pathogenic for humans and highly virulent. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Filoviridae , which is available at www.ictv.global/report/filoviridae.

Minor intron-containing genes (MIGs) account for <2% of all human protein-coding genes and are uniquely dependent on the minor spliceosome for proper excision. Despite their low numbers, we surprisingly found a significant enrichment of MIG-encoded proteins (MIG-Ps) in protein-protein interactomes and host factors of positive-sense RNA viruses, including SARS-CoV-1, SARS-CoV-2, MERS coronavirus, and Zika virus. Similarly, we observed a significant enrichment of MIG-Ps in the interactomes and sets of host factors of negative-sense RNA viruses such as Ebola virus, influenza A virus, and the retrovirus HIV-1. We also found an enrichment of MIG-Ps in double-stranded DNA viruses such as Epstein-Barr virus, human papillomavirus, and herpes simplex viruses. In general, MIG-Ps were highly connected and placed in central positions in a network of human-host protein interactions. Moreover, MIG-Ps that interact with viral proteins were enriched with essential genes. We also provide evidence that viral proteins interact with ancestral MIGs that date back to unicellular organisms and are mainly involved in basic cellular functions such as cell cycle, cell division, and signal transduction. Our results suggest that MIG-Ps form a stable, evolutionarily conserved backbone that viruses putatively tap to invade and propagate in human host cells., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

The 3' untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3' UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3' UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3' UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3' UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts of interest. All authors have submitted the ICMJE forms for disclosure of potential conflicts of interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2023.)

Respiratory syncytial virus (RSV) can cause severe disease especially in infants; however, mechanisms of age-associated disease severity remain elusive. Here, employing human bronchial epithelium models generated from tracheal aspirate-derived basal stem cells of neonates and adults, we investigated whether age regulates RSV-epithelium interaction to determine disease severity. We show that following RSV infection, only neonatal epithelium model exhibited cytopathy and mucus hyperplasia, and neonatal epithelium had more robust viral spread and inflammatory responses than adult epithelium. Mechanistically, RSV-infected neonatal ciliated cells displayed age-related impairment of STAT3 activation, rendering susceptibility to apoptosis, which facilitated viral spread. In contrast, SARS-CoV-2 infection of ciliated cells had no effect on STAT3 activation and was not affected by age. Taken together, our findings identify an age-related and RSV-specific interaction with neonatal bronchial epithelium that critically contributes to severity of infection, and STAT3 activation offers a potential strategy to battle severe RSV disease in infants.

The International Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group continues to prospectively refine the established nomenclature for taxa included in family Filoviridae in an effort to decrease confusion of genus, species, and virus names and to adhere to amended stipulations of the International Code of Virus Classification and Nomenclature (ICVCN). Recently, the genus names Ebolavirus and Marburgvirus were changed to Orthoebolavirus and Orthomarburgvirus, respectively. Additionally, all established species names in family Filoviridae now adhere to the ICTV-mandated binomial format. Virus names remain unchanged and valid. Here, we outline the revised taxonomy of family Filoviridae as approved by the ICTV in April 2023., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories.

Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses., (© 2023. The Author(s).)

Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Inteins (intervening proteins) are polypeptides that interrupt the sequence of other proteins and remove themselves through protein splicing. In this intein-catalyzed reaction, the two peptide bonds surrounding the intein are rearranged to release the intein from the flanking protein sequences, termed N- and C-exteins, which are concurrently joined by a peptide bond. Because of this unique functionality, inteins have proven exceptionally useful in protein engineering. Previous work has demonstrated that heterologous proteins can be inserted within an intein, with both the intein and inserted protein retaining function, allowing for intein-containing genes to coexpress additional coding sequences. Here, we show that a fluorescent protein (ZsGreen) can be inserted within the Pyrococcus horikoshii RadA intein, with the hybrid protein (ZsG-Int) maintaining fluorescence and splicing capability. We used this system to create a recombinant Ebola virus expressing a fluorescent protein. We first tested multiple potential insertion sites for ZsG-Int within individual Ebola virus proteins, identifying a site within the VP30 gene that facilitated efficient intein splicing in mammalian cells while also preserving VP30 function. Next, we successfully rescued a virus containing the ZsG-Int-VP30 fusion protein, which displayed fluorescence in the infected cells. We thus report a new intein-based application for adding reporters to systems without the need to add additional genes. Further, this work highlights a novel reporter design, whereby the reporter is only made if the protein of interest is translated and does not remain fused to the protein of interest.

Liver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a hyperinflammatory state typified by elevated circulating pro-inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID-19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS-CoV-2 binding receptor ACE2. Herein we describe SARS-CoV-2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID-19, demonstrating both heterogeneous ACE2 expression and endothelial damage. In primary endothelial cell cultures, we show that SARS-CoV-2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon-alpha (IFNα) or -beta(β)-two of the main anti-viral cytokines induced in severe SARS-CoV-2 infection-but not significantly by other cytokines (including interleukin 6 and interferon γ/λ). Our findings suggest that the stereotypical anti-viral interferon response may paradoxically facilitate the propagation of COVID-19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID-19., (© 2021. The Author(s).)

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics., (© 2022. The Author(s).)

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 ( ACE2 ), and transmembrane protease serine 2 ( TMPRSS2 ). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness., Competing Interests: The authors have declared that no competing interests exist.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia., (Copyright © 2021 the Author(s). Published by PNAS.)

Several molecular datasets have been recently compiled to characterize the activity of SARS-CoV-2 within human cells. Here we extend computational methods to integrate several different types of sequence, functional and interaction data to reconstruct networks and pathways activated by the virus in host cells. We identify key proteins in these networks and further intersect them with genes differentially expressed at conditions that are known to impact viral activity. Several of the top ranked genes do not directly interact with virus proteins. We experimentally tested treatments for a number of the predicted targets. We show that blocking one of the predicted indirect targets significantly reduces viral loads in stem cell-derived alveolar epithelial type II cells (iAT2s)., Competing Interests: Conflict of interest None.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) usually results in respiratory disease, but extrapulmonary manifestations are of major clinical interest. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Human induced pluripotent stem cell-derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Here, we report that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID-19 as well as its highly variable clinical presentation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system., Competing Interests: Declaration of Interests B.L.W. declares a position as CSO of Aquinnah Pharmaceuticals. A.E. and D.N.K. declare industry funding from Johnson & Johnson, Merck, and Novartis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The spike (S) glycoprotein in the envelope of SARS-CoV-2 is densely glycosylated but the functions of its glycosylation are unknown. Here we demonstrate that S is recognized in a glycan-dependent manner by multiple innate immune receptors including the mannose receptor MR/CD206, DC-SIGN/CD209, L-SIGN/CD209L, and MGL/CLEC10A/CD301. Single-cell RNA sequencing analyses indicate that such receptors are highly expressed in innate immune cells in tissues susceptible to SARS-CoV-2 infection. Binding of the above receptors to S is characterized by affinities in the picomolar range and consistent with S glycosylation analysis demonstrating a variety of N- and O-glycans as receptor ligands. These results indicate multiple routes for SARS-CoV-2 to interact with human cells and suggest alternative strategies for therapeutic intervention., Competing Interests: Competing interest declaration: The authors declare no competing interests.

Bats host a number of viruses that cause severe disease in humans without experiencing overt symptoms of disease themselves. While the mechanisms underlying this ability to avoid sickness are not known, deep sequencing studies of bat genomes have uncovered genetic adaptations that may have functional importance in the antiviral response of these animals. Egyptian rousette bats ( Rousettus aegyptiacus ) are the natural reservoir hosts of Marburg virus (MARV). In contrast to humans, these bats do not become sick when infected with MARV. A striking difference to the human genome is that Egyptian rousettes have an expanded repertoire of IFNW genes. To probe the biological implications of this expansion, we synthesized IFN-ω4 and IFN-ω9 proteins and tested their antiviral activity in Egyptian rousette cells. Both IFN-ω4 and IFN-ω9 showed antiviral activity against RNA viruses, including MARV, with IFN-ω9 being more efficient than IFN-ω4. Using RNA-Seq, we examined the transcriptional response induced by each protein. Although the sets of genes induced by the two IFNs were largely overlapping, IFN-ω9 induced a more rapid and intense response than did IFN-ω4. About 13% of genes induced by IFN-ω treatment are not found in the Interferome or other ISG databases, indicating that they may be uniquely IFN-responsive in this bat., (Copyright © 2020 Pavlovich, Darling, Hume, Davey, Feng, Mühlberger and Kepler.)

Research on filoviruses has historically focused on the highly pathogenic ebola- and marburgviruses. Indeed, until recently, these were the only two genera in the filovirus family. Recent advances in sequencing technologies have facilitated the discovery of not only a new ebolavirus, but also three new filovirus genera and a sixth proposed genus. While two of these new genera are similar to the ebola- and marburgviruses, the other two, discovered in saltwater fishes, are considerably more diverse. Nonetheless, these viruses retain a number of key features of the other filoviruses. Here, we review the key characteristics of filovirus replication and transcription, highlighting similarities and differences between the viruses. In particular, we focus on key regulatory elements in the genomes, replication and transcription strategies, and the conservation of protein domains and functions among the viruses. In addition, using computational analyses, we were able to identify potential homology and functions for some of the genes of the novel filoviruses with previously unknown functions. Although none of the newly discovered filoviruses have yet been isolated, initial studies of some of these viruses using minigenome systems have yielded insights into their mechanisms of replication and transcription. In general, the Cuevavirus and proposed Dianlovirus genera appear to follow the transcription and replication strategies employed by the ebola- and marburgviruses, respectively. While our knowledge of the fish filoviruses is currently limited to sequence analysis, the lack of certain conserved motifs and even entire genes necessitates that they have evolved distinct mechanisms of replication and transcription., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Marburgviruses are closely related to ebolaviruses and cause a devastating disease in humans. In 2012, we published a comprehensive review of the first 45 years of research on marburgviruses and the disease they cause, ranging from molecular biology to ecology. Spurred in part by the deadly Ebola virus outbreak in West Africa in 2013-2016, research on all filoviruses has intensified. Not meant as an introduction to marburgviruses, this article instead provides a synopsis of recent progress in marburgvirus research with a particular focus on molecular biology, advances in animal modeling, and the use of Egyptian fruit bats in infection experiments., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3' and 5' terminal nucleotides because the promoters at the 3' ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3' and 5' terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5' ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5', the 3' termini are variable and are typically 3'-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3' ends. The majority of 3' terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3' terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3'-CCUGU motif regardless of the identity of the 3' terminal nucleotide(s) and of the position of this motif relative to the 3' end. Deletion or mutation of the first residue of the 3'-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3' overhangs. This could be a mechanism to avoid antiviral recognition., Competing Interests: The authors declare no conflict of interest.

Competing Interests: The authors have declared that no competing interests exist

Recently, traces of zoonotic viruses have been discovered in bats and other species around the world, but despite repeated attempts, full viral genomes have not been rescued. The absence of critical genetic sequences from these viruses and the difficulties to isolate infectious virus from specimens prevent research on their pathogenic potential for humans. One example of these zoonotic pathogens is Lloviu virus (LLOV), a filovirus that is closely related to Ebola virus. Here, we established LLOV minigenome systems based on sequence complementation from other filoviruses. Our results show that the LLOV replication and transcription mechanisms are, in general, more similar to ebolaviruses than to marburgviruses. We also show that a single nucleotide at the 3' genome end determines species specificity of the LLOV polymerase. The data obtained here will be instrumental for the rescue of infectious LLOV clones for pathogenesis studies., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ebola virus (EBOV) and Reston virus (RESTV) are members of the Ebolavirus genus which greatly differ in their pathogenicity. While EBOV causes a severe disease in humans characterized by a dysregulated inflammatory response and elevated cytokine and chemokine production, there are no reported disease-associated human cases of RESTV infection, suggesting that RESTV is nonpathogenic for humans. The underlying mechanisms determining the pathogenicity of different ebolavirus species are not yet known. In this study, we dissected the host response to EBOV and RESTV infection in primary human monocyte-derived macrophages (MDMs). As expected, EBOV infection led to a profound proinflammatory response, including strong induction of type I and type III interferons (IFNs). In contrast, RESTV-infected macrophages remained surprisingly silent. Early activation of IFN regulatory factor 3 (IRF3) and NF-κB was observed in EBOV-infected, but not in RESTV-infected, MDMs. In concordance with previous results, MDMs treated with inactivated EBOV and Ebola virus-like particles (VLPs) induced NF-κB activation mediated by Toll-like receptor 4 (TLR4) in a glycoprotein (GP)-dependent manner. This was not the case in cells exposed to live RESTV, inactivated RESTV, or VLPs containing RESTV GP, indicating that RESTV GP does not trigger TLR4 signaling. Our results suggest that the lack of immune activation in RESTV-infected MDMs contributes to lower pathogenicity by preventing the cytokine storm observed in EBOV infection. We further demonstrate that inhibition of TLR4 signaling abolishes EBOV GP-mediated NF-κB activation. This finding indicates that limiting the excessive TLR4-mediated proinflammatory response in EBOV infection should be considered as a potential supportive treatment option for EBOV disease. IMPORTANCE Emerging infectious diseases are a major public health concern, as exemplified by the recent devastating Ebola virus (EBOV) outbreak. Different ebolavirus species are associated with widely varying pathogenicity in humans, ranging from asymptomatic infections for Reston virus (RESTV) to severe disease with fatal outcomes for EBOV. In this comparative study of EBOV- and RESTV-infected human macrophages, we identified key differences in host cell responses. Consistent with previous data, EBOV infection is associated with a proinflammatory signature triggered by the surface glycoprotein (GP), which can be inhibited by blocking TLR4 signaling. In contrast, infection with RESTV failed to stimulate a strong host response in infected macrophages due to the inability of RESTV GP to stimulate TLR4. We propose that disparate proinflammatory host signatures contribute to the differences in pathogenicity reported for ebolavirus species and suggest that proinflammatory pathways represent an intriguing target for the development of novel therapeutics., (Copyright © 2017 American Society for Microbiology.)

This chapter describes the various strategies filoviruses use to escape host immune responses with a focus on innate immune and cell death pathways. Since filovirus replication can be efficiently blocked by interferon (IFN), filoviruses have evolved mechanisms to counteract both type I IFN induction and IFN response signaling pathways. Intriguingly, marburg- and ebolaviruses use different strategies to inhibit IFN signaling. This chapter also summarizes what is known about the role of IFN-stimulated genes (ISGs) in filovirus infection. These fall into three categories: those that restrict filovirus replication, those whose activation is inhibited by filoviruses, and those that have no measurable effect on viral replication. In addition to innate immunity, mammalian cells have evolved strategies to counter viral infections, including the induction of cell death and stress response pathways, and we summarize our current knowledge of how filoviruses interact with these pathways. Finally, this chapter delves into the interaction of EBOV with myeloid dendritic cells and macrophages and the associated inflammatory response, which differs dramatically between these cell types when they are infected with EBOV. In summary, we highlight the multifaceted nature of the host-viral interactions during filoviral infections.

Unlabelled: A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2α phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions., Importance: Although progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Effective inactivation of biosafety level 4 (BSL-4) pathogens is vital in order to study these agents safely. Gamma irradiation is a commonly used method for the inactivation of BSL-4 viruses, which among other advantages, facilitates the study of inactivated yet morphologically intact virions. The reported values for susceptibility of viruses to inactivation by gamma irradiation are sometimes inconsistent, likely due to differences in experimental protocols. We analyzed the effects of common sample attributes on the inactivation of a recombinant vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein and green fluorescent protein. Using this surrogate virus, we found that sample volume and protein content of the sample modulated viral inactivation by gamma irradiation but that air volume within the sample container and the addition of external disinfectant surrounding the sample did not. These data identify several factors which alter viral susceptibility to inactivation and highlight the usefulness of lower biosafety level surrogate viruses for such studies. Our results underscore the need to validate inactivation protocols of BSL-4 pathogens using "worst-case scenario" procedures to ensure complete sample inactivation.