Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.
Ebolavirus/physiology* ; HEK293 Cells ; HeLa Cells ; Humans ; Nucleocapsid Proteins/metabolism* ; RNA, Viral/metabolism* ; Viral Matrix Proteins/metabolism* ; Virion/metabolism* ; Virus Assembly

Influenza C virus is an important respiratory pathogen not only infecting people, but also pigs, dogs, and other animals. Polymerase is central to the replication of influenza C virus and is an important target for studying the mechanism of viral replication. However, there is no commercial monoclonal antibody (MAb) targeting influenza C virus polymerase, which hampers the development of relevant research to some extent. In order to prepare MAb targeting the polymerase basic protein 2 (PB2) of influenza C virus, influenza C virus RNA-dependent RNA polymerase (RdRp, consists of PB1, PB2 and P3) was co-immunoprecipitated with Flag-tagged human acidic nuclear phosphoprotein 32A (huANP32A-Flag) from 293T cells based on the interaction between huANP32A and influenza virus RdRp. The purified RdRp was used as antigen to immunize BALB/c mice. Six positive hybridoma cell lines (7B11-5, 8A4-5, 13D9-6, 8D4-1, 8D4-3, 9F9-4) that stably secrete and recognize PB2 MAb were screened by indirect ELISA and Western blotting. The subtypes of MAb 7B11-5, 8A4-5, 8D4-1 and 8D4-3 antibody were identified as IgG1, the subtypes of MAb 13D9-6 and 9F9-4 were IgG2a and IgG3, respectively. All the light chains of the MAbs were κ chain. A hybridoma cell line 8D4-1 with high titer was further selected to prepare ascites. The titer of mouse ascites antibody was determined to be 1:64 000. Western blotting results showed that the MAb 8D4-1 had a specific immune response with ICV PB2; laser confocal assay showed that the prepared MAb 8D4-1 accurately detected the subcellular localization of PB2 subunits. Moreover, ICV RdRp was highly enriched by ANP32A. The high specific of the prepared PB2 MAb 8D4-1 may facilitate the polymerase detection, structural analysis and mechanism study of influenza C virus.
Animals ; Antibodies, Monoclonal/metabolism* ; Ascites ; Humans ; Influenzavirus C/metabolism* ; Mice ; Nuclear Proteins/metabolism* ; RNA-Binding Proteins ; RNA-Dependent RNA Polymerase/genetics* ; Viral Proteins/metabolism* ; Virus Replication

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.
Adenosine Monophosphate/therapeutic use* ; Alanine/therapeutic use* ; Alveolar Epithelial Cells/virology* ; Antibodies, Neutralizing/therapeutic use* ; COVID-19/virology* ; Down-Regulation ; Drug Discovery ; Human Embryonic Stem Cells/metabolism* ; Humans ; Immunity ; Lipid Metabolism ; Lung/virology* ; RNA, Viral/metabolism* ; SARS-CoV-2/physiology* ; Virus Replication/drug effects*

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
Adult ; Aged ; Aged, 80 and over ; Aging ; genetics ; immunology ; Betacoronavirus ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Lineage ; Chromatin Assembly and Disassembly ; Coronavirus Infections ; immunology ; Cytokine Release Syndrome ; etiology ; immunology ; Cytokines ; biosynthesis ; genetics ; Disease Susceptibility ; Flow Cytometry ; methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Rearrangement ; Humans ; Immune System ; cytology ; growth & development ; immunology ; Immunocompetence ; genetics ; Inflammation ; genetics ; immunology ; Mass Spectrometry ; methods ; Middle Aged ; Pandemics ; Pneumonia, Viral ; immunology ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome ; Young Adult

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.
Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; Betacoronavirus ; drug effects ; physiology ; Binding Sites ; drug effects ; Cell Line ; Coronavirus Infections ; drug therapy ; virology ; Crotonates ; pharmacology ; Cytokine Release Syndrome ; drug therapy ; Drug Evaluation, Preclinical ; Gene Knockout Techniques ; Humans ; Influenza A virus ; drug effects ; Leflunomide ; pharmacology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections ; drug therapy ; Oseltamivir ; therapeutic use ; Oxidoreductases ; antagonists & inhibitors ; metabolism ; Pandemics ; Pneumonia, Viral ; drug therapy ; virology ; Protein Binding ; drug effects ; Pyrimidines ; biosynthesis ; RNA Viruses ; drug effects ; physiology ; Structure-Activity Relationship ; Toluidines ; pharmacology ; Ubiquinone ; metabolism ; Virus Replication ; drug effects

It was widely known that retinoic acid inducible gene I (RIG-I) functions as a cytosolic pattern recognition receptor that initiates innate antiviral immunity by detecting exogenous viral RNAs. However, recent studies showed that RIG-I participates in other various cellular activities by sensing endogenous RNAs under different circumstances. For example, RIG-I facilitates the therapy resistance and expansion of breast cancer cells and promotes T cell-independent B cell activation through interferon signaling activation by recognizing non-coding RNAs and endogenous retroviruses in certain situations. While in hepatocellular carcinoma and acute myeloid leukemia, RIG-I acts as a tumor suppressor through either augmenting STAT1 activation by competitively binding STAT1 against its negative regulator SHP1 or inhibiting AKT-mTOR signaling pathway by directly interacting with Src respectively. These new findings suggest that RIG-I plays more diverse roles in various cellular life activities, such as cell proliferation and differentiation, than previously known. Taken together, the function of RIG-I exceeds far beyond that of a pattern recognition receptor.
Animals ; DEAD Box Protein 58 ; genetics ; metabolism ; Mice ; RNA, Viral ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
Animals ; Connectin/genetics* ; Cricetinae ; Eukaryotic Initiation Factor-4G/genetics* ; Gene Expression Regulation/genetics* ; Gene Silencing ; Growth Hormone/genetics* ; Hepatitis B Surface Antigens/genetics* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Hydro-Lyases/metabolism* ; Male ; Pregnancy-Specific beta 1-Glycoproteins/genetics* ; RNA, Viral/analysis* ; Spermatozoa/virology* ; Trans-Activators/genetics* ; Transcription, Genetic ; Transfection ; Viral Regulatory and Accessory Proteins

No abstract available.
Coronavirus Infections/*diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; Nasopharynx/virology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

From 2013 to 2015, the National Institute of Health, Pakistan, received 1,270 blood samples of suspected dengue cases reported from inpatient and outpatient departments of various hospitals in Khyber Pakhtunkhwa (KPK) province. In this study, we determined the circulating dengue virus (DENV) serotypes using real-time reverse transcriptase (RT)-PCR to understand the serotype-based epidemiology of DENV. All four serotypes (DENV-1 [6%], DENV-2 [33%], DENV-3 [47%], and DENV-4 [0.1%]) were found circulating during the study period. Our findings suggest the need for an active surveillance system coupled with the laboratory diagnosis, especially in the chronic endemic areas of the country. Public awareness programs are needed for effective control and prevention of outbreaks in the future.
Adolescent ; Adult ; Dengue/diagnosis/*epidemiology/virology ; Dengue Virus/genetics/*isolation & purification ; Disease Outbreaks ; Female ; Humans ; Male ; Middle Aged ; Pakistan/epidemiology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Serogroup ; Young Adult

During the 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea, 186 persons were infected, resulting in 38 fatalities. We isolated MERS-CoV from the oropharyngeal sample obtained from a patient of the outbreak. Cytopathic effects showing detachment and rounding of cells were observed in Vero cell cultures 3 days after inoculation of the sample. Spherical virus particles were observed by transmission electron microscopy. Full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade B of MERS-CoV.
Animals ; Cercopithecus aethiops ; Coronavirus Infections/*diagnosis/epidemiology/*virology ; Disease Outbreaks ; Humans ; Microscopy, Electron ; Middle East Respiratory Syndrome Coronavirus/classification/genetics/*isolation & purification/ultrastructure ; Phylogeny ; Polymerase Chain Reaction ; RNA, Viral/analysis/chemistry/metabolism ; Republic of Korea/epidemiology ; Sequence Analysis, RNA ; Vero Cells