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Biology subjects

Orba, Y.

Publications and source records attributed to Orba, Y..

At least 19 recordsLinked to original sources

Reverse genetics system for emerging tick-borne orthonairovirus highlights dispensable N-terminal region of Gn associated with replication in tick vectors

A growing number of emerging and re-emerging tick-borne orthonairoviruses belonging to the Sulina and Tamdy genogroups have recently been identified in association with febrile diseases in East Asia. Yezo virus (YEZV) is one such viruses and is genetically distinct from well-characterized Crimean-Congo hemorrhagic fever virus. Orthonairovirus glycoproteins (Gn and Gc) expressed from a single glycoprotein precursor (GPC) gene mediate interaction with host cells, yet the organization and functions of the YEZV glycoproteins remain largely undefined. To characterize YEZV glycoproteins in the context of infection, we developed a reverse genetics system that enables recovery of recombinant YEZV entirely from cloned cDNAs. The recombinant wild-type virus exhibited growth properties comparable to those of the parental isolate in vitro and maintained pathogenicity in vivo. Proteomic analysis of purified virus particles produced in mammalian cells showed that peptide coverage of GPC-derived products began at residue 69. We therefore used the reverse genetics system to examine the functional importance of the GPC subregion upstream of residue 69. A mutant with a deletion of GPC residues 28 to 68 (rYEZV GPC{Delta}28-68), which retained the predicted signal peptide, was successfully recovered, suggesting that this subregion is dispensable for producing infectious virus. rYEZV GPC{Delta}28-68 propagated at levels comparable to those of the wild-type virus in mammalian cells and exhibited similar pathogenicity in a mouse model. Interestingly, the mutant reached lower viral titers in tick-derived ISE6 cells and in ticks in vivo, suggesting that this N-terminal subregion may contribute to virus replication in ticks. Together, these findings demonstrate the utility of the newly established reverse genetics system for dissecting the functions of the YEZV glycoproteins in mammalian and tick systems.

microbiology↗

Interleukin-34 Promotes Activation of Disease-associated Microglia-like Cells and Attenuates Disease Progression in West Nile Virus Infection

West Nile virus (WNV) is a mosquito-borne orthoflavivirus that causes severe encephalitis, for which no approved antiviral therapies or human vaccines are currently available. Disease-associated microglia (DAM) represent a microglial state associated with neuroprotective functions in neurodegenerative diseases. We previously showed that DAM-like cells are localized in the vicinity of WNV-infected cells in the mouse brain and surmised that these cells may respond to WNV-infected cells. However, the functional significance of this spatial association has since remained unclear. Given the previous reports linking interleukin-34 (IL-34) to the activation of DAM-like cells, we investigated whether IL-34 promotes DAM-like responses and whether these responses are associated with protection against WNV infection. Transcriptomic analysis of IL-34-treated HMC3 human microglial cells revealed upregulation of markers characteristic of DAM, including TREM2, APOE, FABP5, and FTH1. IL-34-treated HMC3 cells suppressed WNV replication in co-culture with WNV-infected SH-SY5Y human neuroblastoma cells apparently in a cell-cell contact-dependent manner, independent of secreted factors. In WNV-infected mice, IL-34 administration improved survival, reduced viral titers, and decreased neuronal apoptosis. IL-34 also increased the abundance of CD11c- and SPP1-positive DAM-like cells, predominantly in the vicinity of WNV-infected cells, in the mouse brain. These findings indicate that IL-34 promotes the activation of DAM-like cells and enhances protective responses in WNV encephalitis. IMPORTANCEWest Nile virus (WNV) is a major cause of viral encephalitis worldwide, with no approved antiviral treatment or human vaccine currently available. Microglia, the resident immune cells of the brain, play key roles in responding to viral infection. However, the mechanism through which specific microglial activation states contribute to protection in viral encephalitis remains poorly understood. In this study, we show that interleukin-34 promotes microglial responses resembling disease-associated microglia (DAM) and is associated with enhanced protection against WNV infection in cell culture and mouse models. The study findings suggest that activation of DAM-like cells may contribute to protective host responses against neurotropic viral infection and provide new insights into the role of specific microglial states in viral encephalitis.

microbiology↗

A surrogate marker of protection confirms the efficacy of an AddaS03-adjuvanted West Nile virus subunit vaccine

West Nile virus (WNV) is the causative agent of fatal West Nile encephalitis. To date, no human vaccine against WNV has been approved. Adjuvants are important for developing effective and affordable vaccines that enhance the immunogenicity and decrease the required antigen doses. In this study, we assessed the efficacy of AddaS03, a synthetic adjuvant analogous to AS03, in a WNV subunit vaccine composed of soluble recombinant envelope protein (sEnv). Using a passive immunization mouse model, we defined the neutralizing antibody titer threshold required for protection against lethal WNV infections and applied this threshold as a surrogate marker to evaluate adjuvant efficacy. AddaS03-adjuvanted formulations elicited markedly higher neutralizing antibody titers compared to Alhydrogel adjuvant 2% (Alhydrogel), even at suboptimal antigen doses, and consistently exceeded the defined protective threshold titer. Moreover, in a sequential challenge mouse model, AddaS03-adjuvanted vaccines completely protected mice from symptomatic WNV infections, whereas Alhydrogel-adjuvanted vaccines failed to confer full protection. Collectively, these findings demonstrate that AddaS03 is a promising adjuvant for WNV subunit vaccine development and highlights the utility of a passive immunization model for defining protective antibody thresholds as a surrogate marker for vaccine evaluation.

microbiology↗

Integrated in vivo and transcriptomic analyses of lethal Oropouche virus infection reveal suppression of pathogenic host responses by antiviral therapy

Oropouche virus (OROV) is an emerging arbovirus responsible for large outbreaks of febrile illness in Central and South America, with increasing reports of severe neurological disease and fatal outcomes. Despite its growing public health impact, no approved antiviral therapies or vaccines are currently available. Here, we show that favipiravir, a broad-spectrum nucleoside analogue, robustly suppresses OROV replication and disease in vivo. In a lethal Syrian hamster model, favipiravir treatment provided complete protection against OROV infection, preventing viral dissemination to peripheral organs and the central nervous system, and remained highly effective when administration was initiated after infection. In contrast, insufficient antiviral control resulted in viral neuroinvasion and fatality. To define host responses associated with OROV pathogenesis and their modulation by antiviral therapy, we performed transcriptomic profiling of liver and brain tissues. OROV infection induced interferon-driven inflammatory programs accompanied by marked disruption of metabolic and tissue homeostatic pathways, whereas these transcriptional signatures were largely abrogated by favipiravir treatment. Together, our findings identify favipiravir as a potent antiviral candidate against OROV and provide the first in vivo, tissue-resolved transcriptomic framework of OROV infection, linking effective viral suppression with the prevention of neuroinvasion and pathogenic host responses. These results highlight antiviral intervention as a viable strategy to mitigate OROV-associated disease and mortality.

microbiology↗

Application of the CPER reverse genetics system for genetic engineering of rabies virus

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for E. coli transformation and cloning of RABV full genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.

microbiology↗

Detection and genetic characterization of pigeon gammacoronavirus

Most members of the genus Gammacoronavirus infect avian hosts, but far fewer viruses have been characterized than in the other coronavirus genera, leaving their diversity largely unclear. Pigeon gammacoronaviruses were previously detected by consensus PCR, but only partial sequences were determined. In this study, we comprehensively analyzed public RNA-seq datasets and reconstructed two nearly complete genomes of pigeon gammacoronaviruses. Molecular evolutionary analyses showed pigeon gammacoronaviruses belong to the subgenus Igacovirus, and their pairwise distances to the members of this subgenus meet the International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria, supporting their designation as a novel species in this subgenus. Notably, although nucleotide sequences are highly conserved gammacoronaviruses, their 3'-genomic region exhibited differences in gene organization among pigeon coronavirus variants. These findings expand our knowledge of the diversity of gammacoronaviruses.

microbiology↗

Simple and quantitative detection of double-stranded RNA as a signature of pan-virus infections using a luciferase-based biosensor

Viral infections produce double-stranded RNA (dsRNA) during replication, which trigger host innate immune responses. Immunoassays using anti-dsRNA antibodies have been widely employed to detect viral dsRNA. In this study, we used a luciferase-based dsRNA biosensor which consists of protein kinase R (PKR)-derived dsRNA binding domains fused to split luciferase subunits. Here, we demonstrate the use of the dsRNA biosensor to measure viral dsRNA in RNA specimens extracted from cells infected with Japanese encephalitis virus (JEV). Moreover, the biosensor reacts to a broad-spectrum of dsRNAs from infection with representatives of various viral families including positive- and negative-sense single-stranded RNA (ssRNA) viruses, dsRNA viruses, and DNA viruses. We validated the specific interaction between the dsRNA biosensor and viral RNA including subgenomic flavivirus RNA (sfRNA) through RNA immunoprecipitation. Additionally, we observed luminescence signals directly from lysates of JEV-infected cells after cell lysis and phase separation with Triton X-114. Finally, we used the biosensor to assess the activity of antiviral compounds. In summary, our results demonstrate that the luciferase-based dsRNA biosensor offers a simple, homogeneous, and high-throughput platform for quantifying viral replication, presenting a promising alternative to antibody-based dsRNA detection methods.

microbiology↗

Snapshots from Cryo-ET of active SARS-CoV-2 virions

Understanding the molecular properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for tackling future outbreaks. Current structural knowledge of the trimeric spike protein relies on truncated recombinant proteins and/or inactivated full-length forms, which may suffer from overstabilization. Here, we apply cryo-electron tomography (cryo-ET) at a Biosafety Level 3 facility to study the virus structure in its native, active state. The virus particles exhibit variable shapes and sizes with diffusible spikes, with the majority in typical prefusion conformations. Notably, we identified unprecedented, a transient open-trimer prefusion states, revealing a hidden flexibility with opened S1 conformation. Subtomogram averaging of the prefusion spikes indicates a loosely packed trimeric architecture that may facilitate the formation of open-trimer state. A cryo-EM map of recombinant Omicron BA.2.75 spike protein further confirms the presence of this loosely packed trimer as a minor conformational state. The observed dynamics uncover conserved cryptic regions that can be targeted for broadly effective vaccines. Structural analysis of active viruses profoundly impacts our understanding of the overlooked fusion mechanism and vaccine, antibody/drug design.

microbiology↗

Combination therapy with oral antiviral and anti-inflammatory drugs improves the efficacy of delayed treatment in severe COVID-19

Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, the treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. Here, we show that the oral antiviral ensitrelvir combined with the anti-inflammatory corticosteroid methylprednisolone has higher therapeutic effects and better outcomes in a delayed dosing model of SARS-CoV-2 infected hamsters compared to the monotherapy with ensitrelvir or methylprednisolone alone. Combination therapy with these drugs improved respiratory conditions and the development of pneumonia in hamsters even when the treatment was started after 2 days post infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulated expressions of genes involved in inflammatory response. Furthermore, we found that the combination treatment is effective in infection with both highly pathogenic delta and circulating omicron variants. Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment. Since both drugs are available as oral medications, this combination therapy could provide a clinical and potent therapeutic option for COVID-19.

microbiology↗

Morphogenesis of bullet-shaped rabies virus particles requires a functional interplay between the viral matrix protein and ESCRT-I component TSG101

Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host ESCRT (endosomal sorting complex required for transport) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection, and specifically the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a siRNA screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late-domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. ImportanceEnveloped-viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT (endosomal sorting complex required for transport) complexes. Some enveloped-viruses utilize their late (L)-domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped-virions, but the underlying molecular mechanisms involved remain elusive. Herein, we showed that TSG101, one of ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

microbiology↗

2-Thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes significant morbidity and mortality worldwide, seriously impacting not only human health but also the global economy. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these and many other viruses have been approved, they continue to be responsible for large-scale epidemics and global pandemics. Thus, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral nucleoside analogue that exhibited antiviral activity against SARS-CoV-2 and its variants of concern, including the Delta and Omicron variants, as well as a number of other positive-sense single-stranded RNA (ssRNA+) viruses, including DENV. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with SARS-CoV-2 or DENV in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against SARS-CoV-2 and DENV but other ssRNA+ viruses.

microbiology↗

Discovery of Chlorofluoroacetamide-Based Covalent Inhibitors for SARS-CoV-2 3CL Protease

The pandemic of coronavirus disease 2019 (COVID-19) has urgently necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report the new class of covalent inhibitors for 3CLpro possessing chlorofluoroacetamide (CFA) as a cysteine reactive warhead. Based on the aza-peptide scaffold, we synthesized the series of CFA derivatives in enantiopure form and evaluated their biochemical efficiencies. The data revealed that 8a (YH-6) with R configuration at the CFA unit strongly blocks the SARS-CoV-2 replication in the infected cells and this potency is comparable to that of nirmatrelvir. The X-ray structural analysis shows that 8a (YH-6) forms a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and sufficient pharmacokinetics property of 8a (YH-6) suggest its potential as a lead compound for treatment of COVID-19.

biochemistry↗

Virological characteristics of SARS-CoV-2 BA.2 variant

Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref1, 2), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.

microbiology↗

Oral administration of S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and accelerates recovery from clinical aspects of COVID-19

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622, a novel inhibitor of SARS-CoV-2 main protease (Mpro, also known as 3C-like protease), decreases viral load and ameliorates the disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to sub-micromolar concentrations in cells. Oral administration of S-217622 demonstrated eminent pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern (VOCs), including the highly pathogenic Delta variant and the recently emerged Omicron variant. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase II/III clinical trial, possesses remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

microbiology↗

Discovery of S-217622, a Non-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral non-covalent, non-peptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug-design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel non-covalent inhibitor could be a potential oral agent for treating COVID-19.

biochemistry↗

LIGHTHOUSE illuminates therapeutics for a variety of diseases including COVID-19

One of the bottlenecks in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs for given therapeutic targets. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified novel therapeutics for cancer, lifestyle-related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. We envision that LIGHTHOUSE will bring about a paradigm shift in translational medicine, providing a bridge from bench side to bedside.

systems biology↗

SARS-CoV-2 bearing a mutation at the S1/S2 cleavage site exhibits attenuated virulence and confers protective immunity

Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by host furin protease. Proteolytic cleavage activates the spike protein and influences both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogensis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited a low growth property. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, indicating the attenuated variant nature of S gene mutants. We reproduced the attenuated growth of S gene mutants in primary differenciated human airway epithelial cells. This transient infection was enough to induce protective neutralizing antibodies crossreacting with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of SARS-CoV-2 and highlights the potential benefits of S gene mutants as potential immunogens.

microbiology↗

Combinatorial approach with mass spectrometry and lectin microarray dissected glycoproteomic features of virion-derived spike protein of SARS-CoV-2

The COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2, has a global impact on public health. Since glycosylation of the viral envelope glycoproteins is known to be deeply associated with their immunogenicity, intensive studies on the glycans of its major glycoprotein, S protein, have been conducted. Nevertheless, the detailed site-specific glycan compositions of virion-associated S protein have not yet been clarified. Here, we conducted intensive glycoproteomic analyses of SARS-CoV-2 S protein using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarray. Using our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, we identified 1,759 site-specific glycan compositions. The most frequent was HexNAc:Hex:Fuc:NeuAc:NeuGc = 6:6:1:0:0, suggesting a tri-antennary N-glycan terminating with LacNAc and having bisecting GlcNAc and a core fucose, which was found in 20 of 22 glycosylated sites. The subsequent lectin microarray analysis emphasized intensive outer arm fucosylation of glycans, which efficiently complemented the glycoproteomic features. The present results illustrate the high-resolution glycoproteomic features of SARS-CoV-2 S protein and significantly contribute to vaccine design, as well as the understanding of viral protein synthesis.

molecular biology↗