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Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5' triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5' triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5' monophosphate-specific exonuclease indicates that the RIG-I-activating 5' triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5' end as a strategy of viruses to evade the innate immune response.

In April 2023, 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 one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article examines the role of toll-like receptor 4 (TLR4) in damaging inflammatory responses during acute viral infections. Topics discussed include the ligands of TLR4, activation of viruses that induce an inflammatory response during acute infection through TLR4, and the therapeutic potential of TLR4. The role of over-stimulation of TLR4 in excessive inflammatory response that is damaging to the host and its benefits to the host by establishing a protective immune response are explained.

Protein kinase R (PKR) is a key antiviral protein involved in sensing and restricting viral infections. Here we analyzed the ability of Marburg virus (MARV) viral protein 35 (VP35) to inhibit PKR activation in human and bat cells. Similar to the related Ebola and Lloviu viruses, MARV VP35 was able to inhibit PKR activation in 293T cells. In contrast, we found that MARV VP35 did not inhibit human or bat PKR activation in human glioblastoma U-251-MG cells or a Rousettus aegyptiacus cell line. Additional experiments revealed that PACT, a known PKR regulator, was insufficient to rescue the ability of VP35 to inhibit PKR activation in these cells. Taken together, this study indicates that the ability of VP35 to inhibit PKR is cell type specific, potentially explaining discrepancies between the ability of filoviruses to potently block innate immune responses, and the high levels of interferon and interferon-stimulated genes observed in filovirus patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The highly pathogenic filoviruses, Marburg and Ebola virus, belong to the nonsegmented negative-sense RNA viruses of the order Mononegavirales. The mode of replication and transcription is similar for these viruses. On one hand, the negative-sense RNA genome serves as a template for replication, to generate progeny genomes, and, on the other hand, for transcription, to produce mRNAs. Despite the similarities in the replication/transcription strategy, filoviruses have evolved structural and functional properties that are unique among the nonsegmented negative-sense RNA viruses. Moreover, there are also striking differences in the replication and transcription mechanisms of Marburg and Ebola virus. This includes nucleocapsid formation, the structure of the genomic replication promoter, the protein requirement for transcription and the use of mRNA editing. In this article, the current knowledge of the replication and transcription strategy of Marburg and Ebola virus is reviewed, with focus on the observed differences. [ABSTRACT FROM AUTHOR]

Ebola virus VP35 contains a C-terminal cluster of basic amino acids required for double-stranded RNA (dsRNA) binding and inhibition of interferon regulatory factor 3 (IRF3). VP35 also blocks protein kinase R (PKR) activation; however, the responsible domain has remained undefined. Here we show that the IRF inhibitory domain of VP35 mediates the inhibition of PKR and enhances the synthesis of coexpressed proteins. In contrast to dsRNA binding and IRF inhibition, alanine substitutions of at least two basic amino acids are required to abrogate PKR inhibition and enhanced protein expression. Moreover, we show that PKR activation is not only blocked but reversed by Ebola virus infection. [ABSTRACT FROM AUTHOR]