Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection.

Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/ß-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of ß-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, ß-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted ß-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This ß-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by ß-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases.

Maternal SARS-CoV-2 infection in pregnancy disrupts gene expression in Hofbauer cells with limited impact on cytotrophoblasts.

BACKGROUND: Hofbauer cells (HBCs) and cytotrophoblasts (CTBs) are major cell populations in placenta. The indirect impact of maternal SARS-CoV-2 disease on these cells that are not directly infected has not been extensively studied. Herein, we profiled gene expression in HBCs and CTBs isolated from placentae of recovered pregnant subjects infected with SARS-CoV-2 during all trimesters of pregnancy, placentae from subjects with active infection, SARS-CoV-2 vaccinated subjects, and those who were unexposed to the virus. METHODS: Placentae were collected within 4 h post-delivery and membrane-free tissues were enzymatically digested for the isolation of HBCs and CTBs. RNA extracted from HBCs and CTBs were sequenced using 150bp paired-end reads. Differentially expressed genes (DEGs) were identified by DESeq2 package in R and enriched in GO Biological Processes, KEGG Pathway, Reactome Gene Sets, Hallmark Gene Sets, and Canonical Pathways. Protein-protein interactions among the DEGs were modelled using STRING and BioGrid. RESULTS: Pregnant subjects (n = 30) were recruited and categorized into six groups: infected with SARS-CoV-2 in i) the first (1T, n = 4), ii) second (2T, n = 5), iii) third (3T, n = 5) trimester, iv) tested positive at delivery (Delivery, n = 5), v) never infected (Control, n = 6), and vi) fully mRNA-vaccinated by delivery (Vaccinated, n = 5). Compared to the Control group, gene expression analysis showed that HBCs from infected subjects had significantly altered gene expression profiles, with the 2T group having the highest number of DEGs (1,696), followed by 3T and 1T groups (1,656 and 958 DEGs, respectively). These DEGs were enriched for pathways involved in immune regulation for host defense, including production of cytokines, chemokines, antimicrobial proteins, ribosomal assembly, neutrophil degranulation inflammation, morphogenesis, and cell migration/adhesion. Protein-protein interaction analysis mapped these DEGs with oxidative phosphorylation, translation, extracellular matrix organization, and type I interferon signaling. Only 95, 23, and 8 DEGs were identified in CTBs of 1T, 2T, and 3T groups, respectively. Similarly, 11 and 3 DEGs were identified in CTBs and HBCs of vaccinated subjects, respectively. Reassuringly, mRNA vaccination did not induce an inflammatory response in placental cells. CONCLUSIONS: Our studies demonstrate a significant impact of indirect SARS-CoV-2 infection on gene expression of inner mesenchymal HBCs, with limited effect on lining CTB cells isolated from pregnant subjects infected and recovered from SARS-CoV-2. The pathways associated with these DEGs identify potential targets for therapeutic intervention.

Vertical Transmission of SARS-CoV-2-Specific Antibodies and Cytokine Profiles in Pregnancy.

Despite intensive characterization of immune responses after COVID-19 infection and vaccination, research examining protective correlates of vertical transmission in pregnancy are limited. Herein, we profiled humoral and cellular characteristics in pregnant women infected or vaccinated at different trimesters and in their corresponding newborns. We noted a significant correlation between spike S1-specific IgG antibody and its RBD-ACE2 blocking activity (receptor-binding domain-human angiotensin-converting enzyme 2) in maternal and cord plasma (P < .001, R > 0.90). Blocking activity of spike S1-specific IgG was significantly higher in pregnant women infected during the third trimester than the first and second trimesters. Elevated levels of 28 cytokines/chemokines, mainly proinflammatory, were noted in maternal plasma with infection at delivery, while cord plasma with maternal infection 2 weeks before delivery exhibited the emergence of anti-inflammatory cytokines. Our data support vertical transmission of protective SARS-CoV-2-specific antibodies. This vertical antibody transmission and the presence of anti-inflammatory cytokines in cord blood may offset adverse outcomes of inflammation in exposed newborns.

Macacine alphaherpesvirus 1 (B Virus) Infection in Humans, Japan, 2019.

Two human patients with Macacine alphaherpesvirus 1 infection were identified in Japan in 2019. Both patients had worked at the same company, which had a macaque facility. The rhesus-genotype B virus genome was detected in cerebrospinal fluid samples from both patients.

Efficacy and viral dynamics of tecovirimat in patients with MPOX: A multicenter open-label, double-arm trial in Japan.

INTRODUCTION: Tecovirimat's application in treating mpox remains under-researched, leaving gaps in clinical and virological understanding. METHODS: The Tecopox study in Japan evaluated the efficacy and safety of tecovirimat in patients with smallpox or mpox, who were divided into oral tecovirimat and control groups. Patients with mpox enrolled between June 28, 2022, and April 30, 2023, were included. Demographic and clinical details along with blood, urine, pharyngeal swab, and skin lesion samples were gathered for viral analysis. A multivariable Tobit regression model was employed to identify factors influencing prolonged viral detection. RESULTS: Nineteen patients were allocated to the tecovirimat group, and no patients were allocated to the control group. The median age was 38.5 years, and all patients were males. Ten patients (52.6%) were infected with human immunodeficiency virus (HIV). Sixteen patients (84.2%) had severe disease. Nine of the 15 patients (60.0%) (four patients withdrew before day 14) had negative PCR results for skin lesion specimens 14 days after inclusion. The mortality rates were 0% on days 14 and 30. No severe adverse events were reported. HIV status and the number of days from symptom onset to tecovirimat administration were associated with lower Ct values (p = 0.027 and p < 0.001, respectively). The median number of days when PCR testing did not detect the mpox virus in each patient was 19.5 days. CONCLUSION: Early tecovirimat administration might reduce viral shedding duration, thereby mitigating infection spread. Moreover, patients infected with HIV showed prolonged viral shedding, increasing the transmission risk compared to those without HIV.

Potential Anti-Mpox Virus Activity of Atovaquone, Mefloquine, and Molnupiravir, and Their Potential Use as Treatments.

BACKGROUND: Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling. METHODS: We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment. RESULTS: Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 µM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations. CONCLUSIONS: These data suggest that atovaquone would be a potential candidate for treating mpox.

Role of VP30 Phosphorylation in Ebola Virus Nucleocapsid Assembly and Transport.

Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. IMPORTANCE Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.

Proteomic Signature of Host Response to SARS-CoV-2 Infection in the Nasopharynx.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a global health pandemic. COVID-19 severity ranges from an asymptomatic infection to a severe multiorgan disease. Although the inflammatory response has been implicated in the pathogenesis of COVID-19, the exact nature of dysregulation in signaling pathways has not yet been elucidated, underscoring the need for further molecular characterization of SARS-CoV-2 infection in humans. Here, we characterize the host response directly at the point of viral entry through analysis of nasopharyngeal swabs. Multiplexed high-resolution MS-based proteomic analysis of confirmed COVID-19 cases and negative controls identified 7582 proteins and revealed significant upregulation of interferon-mediated antiviral signaling in addition to multiple other proteins that are not encoded by interferon-stimulated genes or well characterized during viral infections. Downregulation of several proteasomal subunits, E3 ubiquitin ligases, and components of protein synthesis machinery was significant upon SARS-CoV-2 infection. Targeted proteomics to measure abundance levels of MX1, ISG15, STAT1, RIG-I, and CXCL10 detected proteomic signatures of interferon-mediated antiviral signaling that differentiated COVID-19-positive from COVID-19-negative cases. Phosphoproteomic analysis revealed increased phosphorylation of several proteins with known antiviral properties as well as several proteins involved in ciliary function (CEP131 and CFAP57) that have not previously been implicated in the context of coronavirus infections. In addition, decreased phosphorylation levels of AKT and PKC, which have been shown to play varying roles in different viral infections, were observed in infected individuals relative to controls. These data provide novel insights that add depth to our understanding of SARS-CoV-2 infection in the upper airway and establish a proteomic signature for this viral infection.

The combination of levodopa with levodopa-metabolizing enzyme inhibitors prevents severe fever with thrombocytopenia syndrome virus infection in vitro more effectively than single levodopa.

Severe fever with thrombocytopenia syndrome is a hemorrhagic fever caused by a tick-borne infection. The causative agent, Dabie bandavirus, is also called the severe fever with thrombocytopenia syndrome virus (SFTSV). Ogawa et al. (2022) reported that levodopa, an antiparkinsonian drug with an o-dihydroxybenzene backbone, which is important for anti-SFTSV activity, inhibited SFTSV infection. Levodopa is metabolized by dopa decarboxylase (DDC) and catechol-O-methyltransferase (COMT) in vivo. We evaluated the anti-SFTSV efficacy of two DDC inhibitors, benserazide hydrochloride and carbidopa, and two COMT inhibitors, entacapone and nitecapone, which also have an o-dihydroxybenzene backbone. Only DDC inhibitors inhibited SFTSV infection with pretreatment of the virus (half-maximal inhibitory concentration [IC50]: 9.0-23.6 µM), whereas all the drugs inhibited SFTSV infection when infected cells were treated (IC50: 21.3-94.2 µM). Levodopa combined with carbidopa and/or entacapone inhibited SFTSV infection in both conditions: pretreatment of the virus (IC50: 2.9-5.8 µM) and treatment of infected cells (IC50: 10.7-15.4 µM). The IC50 of levodopa in the above-mentioned study for pretreatment of the virus and treatment of infected cells were 4.5 and 21.4 µM, respectively. This suggests that a synergistic effect was observed, especially for treatment of infected cells, although the effect is unclear for pretreatment of the virus. This study demonstrates the anti-SFTSV efficacy of levodopa-metabolizing enzyme inhibitors in vitro. These drugs may increase the time for which the levodopa concentration is maintained in vivo. The combination of levodopa and levodopa-metabolizing enzyme inhibitors might be a candidate for drug repurposing.

Treatment with tecovirimat of the first two cases of monkeypox in Japan.

Outbreaks of monkeypox in Europe and North America have been reported since May 2022. At the end of July, we encountered the first two cases of monkeypox diagnosed in Japan. Case 1 was a white man who traveled to Spain where he had sexual intercourse with men. He presented to our hospital with fever, rash, and tiredness, and was diagnosed with monkeypox based on positive PCR test results from the skin lesions. He was admitted to our hospital, received tecovirimat 600 mg twice daily, and was discharged on day 15. Case 2 involved a Japanese man who visited us because of fatigue, muscle pain, headache, and oral ulcers. He was living in New York and traveled to Japan one day before presentation. He had experienced sexual intercourse with men four times during the previous month. The patient was diagnosed with monkeypox based on positive PCR results from the blood. He was admitted to our hospital, received tecovirimat 600 mg twice daily, and was discharged on day 14. These were the first two cases of monkeypox diagnosed in Japan. Based on their history and epidemiology, the viruses seem to have been imported from Europe and North America, respectively. After initiation of tecovirimat, both patients showed mild symptoms and immediate disappearance of viral DNA. The second case was notable for being diagnosed without skin rash. Our report suggests that tecovirimat could decrease the viral load rapidly, and that our prompt diagnosis contributed to the prevention of a monkeypox outbreak in Japan.

Ebolavirus polymerase uses an unconventional genome replication mechanism.

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.

Strengthening the Interaction of the Virology Community with the International Committee on Taxonomy of Viruses (ICTV) by Linking Virus Names and Their Abbreviations to Virus Species.

The International Committee on Taxonomy of Viruses (ICTV) is tasked with classifying viruses into taxa (phyla to species) and devising taxon names. Virus names and virus name abbreviations are currently not within the ICTV's official remit and are not regulated by an official entity. Many scientists, medical/veterinary professionals, and regulatory agencies do not address evolutionary questions nor are they concerned with the hierarchical organization of the viral world, and therefore, have limited use for ICTV-devised taxa. Instead, these professionals look to the ICTV as an expert point source that provides the most current taxonomic affiliations of viruses of interests to facilitate document writing. These needs are currently unmet as an ICTV-supported, easily searchable database that includes all published virus names and abbreviations linked to their taxa is not available. In addition, in stark contrast to other biological taxonomic frameworks, virus taxonomy currently permits individual species to have several members. Consequently, confusion emerges among those who are not aware of the difference between taxa and viruses, and because certain well-known viruses cannot be located in ICTV publications or be linked to their species. In addition, the number of duplicate names and abbreviations has increased dramatically in the literature. To solve this conundrum, the ICTV could mandate listing all viruses of established species and all reported unclassified viruses in forthcoming online ICTV Reports and create a searchable webpage using this information. The International Union of Microbiology Societies could also consider changing the mandate of the ICTV to include the nomenclature of all viruses in addition to taxon considerations. With such a mandate expansion, official virus names and virus name abbreviations could be catalogued and virus nomenclature could be standardized. As a result, the ICTV would become an even more useful resource for all stakeholders in virology.

Taxonomy of the order Bunyavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Taxonomy of the order Bunyavirales: second update 2018.

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Ebolaviruses Associated with Differential Pathogenicity Induce Distinct Host Responses in Human Macrophages.

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.

Small Animal Models for Studying Filovirus Pathogenesis.

Filovirus small animal disease models have so far been developed in laboratory mice, guinea pigs, and hamsters. Since immunocompetent rodents do not exhibit overt signs of disease following infection with wild-type filoviruses isolated from humans, rodent models have been established using adapted viruses produced through sequential passage in rodents. Rodent-adapted viruses target the same cells/tissues as the wild-type viruses, making rodents invaluable basic research tools for studying filovirus pathogenesis. Moreover, comparative analyses using wild-type and rodent-adapted viruses have provided beneficial insights into the molecular mechanisms of pathogenicity and acquisition of species-specific virulence. Additionally, wild-type filovirus infections in immunodeficient rodents have provided a better understanding of the host factors required for resistance to filovirus infection and of the immune response against the infection. This chapter provides comprehensive information on the filovirus rodent models and rodent-adapted filoviruses. Specifically, we summarize the clinical and pathological features of filovirus infections in all rodent models described to date, including the recently developed humanized and collaborative cross (CC) resource recombinant inbred (RI) intercrossed (CC-RIX) mouse models. We also cover the molecular determinants responsible for adaptation and virulence acquisition in a number of rodent-adapted filoviruses. This chapter clearly defines the characteristic and advantages/disadvantages of rodent models, helping to evaluate the practical use of rodent models in future filovirus studies.

Taxonomy of the order Mononegavirales: update 2018.

In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018.

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Maguari Virus Associated with Human Disease.

Despite the lack of evidence for symptomatic human infection with Maguari virus (MAGV), its close relation to Cache Valley virus (CVV), which does infect humans, remains a concern. We sequenced the complete genome of a MAGV-like isolate (OBS6657) obtained from a febrile patient in Pucallpa, Ucayali, Peru, in 1998. To facilitate its classification, we generated additional full-length sequences for the MAGV prototype strain, 3 additional MAGV-like isolates, and the closely related CVV (7 strains), Tlacotalpan (1 strain), Playas (3 strains), and Fort Sherman (1 strain) viruses. The OBS6657 isolate is similar to the MAGV prototype, whereas 2 of the other MAGV-like isolates are located on a distinct branch and most likely warrant classification as a separate virus species and 1 is, in fact, a misclassified CVV strain. Our findings provide clear evidence that MAGV can cause human disease.

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions.

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.