Ebihara, Hideki

Uncontrolled systemic inflammatory responses are a critical pathological feature of fatal Ebola virus (EBOV) infection. While some inflammatory responses may originate from mononuclear phagocytes (MNPs), nonimmune cells vastly outnumber MNPs and may be an important source of inflammation. Here, we demonstrated that highly virulent EBOV induced a high and sustained pro-inflammatory response compared to less virulent ebolaviruses in non-MNPs through TLR4-independent NF-κB activation. We identified the EBOV matrix protein VP40 as a potent activator of NF-κB in non-MNPs, whose intrinsic inflammatory activation ability is higher than VP40 proteins from less virulent ebolaviruses. This suggests that VP40 is a virulence determinant inducing distinct degrees of pro-inflammatory responses among ebolaviruses. Mechanistically, VP40 activated the NF-κB signaling pathway, primarily via TNFR1 using a ligand-independent mechanism. These findings reveal mechanisms that may drive systemic inflammation and promote EBOV pathogenesis, suggesting potential therapeutic strategies to mitigate immune dysregulation in severe EBOV infections.

ABSTRACT Thogotoviruses are arthropod-borne viruses belonging to the genus Thogotovirus within the family Orthomyxoviridae . Like other orthomyxoviruses, such as the influenza A viruses (IAV), thogotoviruses replicate in the nucleus. As a result, progeny viral ribonucleoprotein complexes (vRNPs) must be exported to the cytoplasm prior to virion assembly and budding at the plasma membrane. In IAV, this export depends on binding of the viral nuclear export protein (NEP) to the cellular exportin chromosomal maintenance 1 (CRM1). In contrast, the mechanism of vRNP nuclear export, including identification of a protein with NEP functionality, has not been characterized for any thogotoviruses. Here, we characterized vRNP nuclear export in Dhori virus (DHOV), a prototypic member of the Thogotovirus genus. DHOV replication and nuclear export of the viral nucleoprotein were inhibited by the canonical CRM1 inhibitor leptomycin B (LMB), suggesting that DHOV vRNP export also utilizes CRM1. Interestingly, LMB treatment led to nuclear retention of the DHOV matrix (M) protein in both infected and transfected cells. Using a mammalian two-hybrid system, we found that DHOV M interacts with CRM1 through a nuclear export sequence (NES) located between amino acids 111 and 128. Mutation of hydrophobic residues within this NES reduced M-CRM1 interaction, abolished the NES phenotype when fused to a fluorescent protein, and impaired rescue of recombinant DHOV by reverse genetics. Together, our results reveal that DHOV vRNP nuclear export is CRM1-dependent and mediated by the M protein rather than a dedicated NEP-like protein, providing the first mechanistic insight into vRNP egress in the genus Thogotovirus . IMPORTANCE Dhori virus (DHOV) is a pathogenic tick-borne virus in the genus Thogotovirus , in the family Orthomyxoviridae. Despite evidence of DHOV exposure in various mammals, including humans, its basic biology is not well understood. We investigated how DHOV’s progeny genome and protein complexes—viral ribonucleoprotein complexes (vRNPs)—are transported out of the nucleus. Our findings show that DHOV, like the influenza viruses, uses the cellular protein chromosomal maintenance protein 1 (CRM1) for vRNP export. We found that chemically inhibiting CRM1 completely blocked DHOV vRNP export, preventing the production of progeny viruses from infected cells. Screening of all known DHOV proteins revealed that the matrix protein, which forms the virus’ scaffold, interacted with CRM1, suggesting it may link CRM1 to the vRNPs. These results advance our understanding of DHOV replication and suggest that chemically inhibiting vRNP export could be a way to treat thogotovirus infections.

Ebola virus (EBOV) causes severe disease outbreaks in humans with high case fatality rates. EBOV requires adaptation to cause lethal disease in mice by acquiring single mutations in both the nucleoprotein (NP) and VP24 genes. As an attempt to model mouse-adapted EBOV (MA-EBOV), we engineered novel pentacistronic minigenomes (5xMG) containing a reporter gene, VP40, and glycoprotein genes as well as the NP and VP24 genes from either EBOV or MA-EBOV. The 5xMGs were constructed and optimized, and the produced transcription- and replication-competent virus-like particles (trVLPs) were demonstrated to infect several cell lines. Introduction of the mouse-adaptation mutations did not significantly impact the replication and transcription of the 5xMG or the relative infectivity of the trVLPs in vitro. This work demonstrates the development of the 5xMG system as a new versatile tool to study EBOV biology.

Abstract 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.

Abstract Background Despite extensive studies of human immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) vaccination, research examining protective correlates of vertical transmission following maternal exposure in pregnancy remain limited. Here, we characterized antibody and cytokine responses in maternal and cord blood following infection or vaccination at various timepoints during gestation. Methods Spike S1 protein-specific binding antibodies and antibodies capable of blocking the interaction between the receptor binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) were measured in maternal and cord blood by ELISA. Serum concentrations of 74 cytokines/chemokines were measured by multiplex assay. Humoral responses and cytokine levels from matched maternal and fetal cord sera were compared and examined for potential correlations. Results We observed a highly significant correlation between Spike S1-specific antibody titer and RBD-ACE2 blocking antibody activity between maternal and fetal cord serum (p < 2.2e-16, R > 0.90). Blocking antibody activity was significantly higher for mothers infected during the 3rd trimester compared to earlier trimesters; however, vaccinated mothers developed and transferred higher antibody titers with greater RBD-ACE2 blocking antibody activity to their neonates than infected mothers. Furthermore, vaccine-induced Spike S1 IgG transfer ratios (fetal cord/maternal) were significantly higher than those induced by infection (p = 0.002). Multiplex assay showed significantly elevated levels of 33 cytokines/chemokines, mainly pro-inflammatory in infected maternal serum samples, while the paired fetal cord samples exhibited an anti-inflammatory cytokine predominance. Conclusion Our data support selective vertical transmission of potentially protective humoral responses against SARS-CoV-2, especially following vaccination in the 3rd trimester. The anti-inflammatory cytokine predominance in cord blood that persists despite maternal SARS-CoV-2 infection may offset the adverse outcomes of inflammation in pregnancy for the neonate. Disclosures All Authors: No reported disclosures.

We reported the genetic evidence of circulating hantaviruses from small mammals captured in a chronic kidney disease of unknown etiology (CKDu) hotspot area of Sri Lanka. The high seroprevalence of anti-hantavirus antibodies against Thailand orthohantavirus (THAIV) has been reported among CKDu patients and rodents in Sri Lankan CKDu hotspots. We captured 116 small mammals from CKDu endemic regions in the Polonnaruwa District of Sri Lanka. Seven animals (five out of 11 Mus booduga and two out of 99 Rattus rattus) were PCR-positive for the hantavirus. A rat-borne sequence was grouped with a THAIV-like Anjozorobe virus. In contrast, Mus-borne sequences belonged to the THAIV lineage, suggesting a novel orthohantavirus species according to the phylogenetic analyses and whole-genome comparisons. Our genetic evidence indicates the presence of two THAIV-related viruses circulating in this CKDu endemic area, suggesting a basis for further investigations to identify the infectious virus in patients with CKDu and the CKDu induction mechanism of these viruses.

COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (>20% located in United States) and killed more than 3.3 million people around the world (>20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors—S/Ace2, Tmprss2, Cathepsins L and K, and Mpro—to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments.

Context.— Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription–polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). Objective.— To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. Design.— Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. Results.— Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (κ = 0.75). Interobserver reliability was almost perfect for IHC (κ = 0.91) and fair to moderate for RNAish (κ = 0.38–0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P = .03, Pearson correlation = −0.65) and were more likely to be positive by RNAish (P = .02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. Conclusions.— Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

Lymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus. The function of untranslated regions (UTRs) of the LCMV genome has not been well studied except for the extreme 19 nucleotide residues of both the 5′ and 3′ termini. There are internal UTRs composed of 58 and 41 nucleotide residues in the 5′ and 3′ UTRs, respectively, in the LCMV S segment. Their functional roles have yet to be elucidated. In this study, reverse genetics and minigenome systems were established for LCMV strain WE and the function of these regions were analyzed. It was revealed that nucleotides 20–40 and 20–38 located downstream of the 19 nucleotides in the 5′ and 3′ termini, respectively, were involved in viral genome replication and transcription. Furthermore, it was revealed that the other internal UTRs (nucleotides 41–77 and 39–60 in the 5′ and 3′ termini, respectively) in the S segment were involved in virulence in vivo, even though these regions did not affect viral growth capacity in Vero cells. The introduction of LCMV with mutations in these regions attenuates the virus and may enable the production of LCMV vaccine candidates.