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Viruses Aug 2023There is no specific chemotherapy approved for the treatment of pathogenic arenaviruses that cause severe hemorrhagic fever (HF) in the population of endemic regions in...
There is no specific chemotherapy approved for the treatment of pathogenic arenaviruses that cause severe hemorrhagic fever (HF) in the population of endemic regions in America and Africa. The present study reports the effects of the natural flavonoid quercetin (QUER) on the infection of A549 and Vero cells with Junín virus (JUNV), agent of the Argentine HF. By infectivity assays, a very effective dose-dependent reduction of JUNV multiplication was shown by cell pretreatment at 2-6 h prior to the infection at non-cytotoxic concentrations, with 50% effective concentration values in the range of 6.1-7.5 µg/mL. QUER was also active by post-infection treatment but with minor efficacy. Mechanistic studies indicated that QUER mainly affected the early steps of virus adsorption and internalization in the multiplication cycle of JUNV. Treatment with QUER blocked the phosphorylation of Akt without changes in the total protein expression, detected by Western blot, and the consequent perturbation of the PI3K/Akt pathway was also associated with the fluorescence redistribution from membrane to cytoplasm of TfR1, the cell receptor recognized by JUNV. Then, it appears that the cellular antiviral state, induced by QUER treatment, leads to the prevention of JUNV entry into the cell.
Topics: Chlorocebus aethiops; Animals; Quercetin; Flavonoids; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Vero Cells; Arenaviridae Infections; Arenavirus
PubMed: 37632083
DOI: 10.3390/v15081741 -
Frontiers in Immunology 2023Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit...
INTRODUCTION
Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed.
METHODS
Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8 T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens.
RESULTS
Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8 T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer CD8 T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8 T cells that were GFP was <1%. These data show that a significant percentage of viral-specific memory CD8 T cells expressed T cell receptors that also recognized alloantigens . Notably, the frequency of cross-reactive CD8 T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity.
DISCUSSION
In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.
Topics: Mice; Animals; CD8-Positive T-Lymphocytes; Mice, Inbred C57BL; Lymphocytic choriomeningitis virus; Virus Diseases; Receptors, Antigen, T-Cell; Isoantigens; Allografts
PubMed: 38143762
DOI: 10.3389/fimmu.2023.1287546 -
Scientific Reports Aug 2023The World Health Organization's R&D Blueprint list of priority diseases for 2022 includes Lassa fever, signifying the need for research and development in emergency...
The World Health Organization's R&D Blueprint list of priority diseases for 2022 includes Lassa fever, signifying the need for research and development in emergency contexts. This disease is caused by the arenavirus Lassa virus (LASV). Being an enveloped virus, LASV should be susceptible to a variety of microbicidal actives, although empirical data to support this expectation are needed. We evaluated the virucidal efficacy of sodium hypochlorite, ethanol, a formulated dual quaternary ammonium compound, an accelerated hydrogen peroxide formulation, and a p-chloro-m-xylenol formulation, per ASTM E1052-20, against LASV engineered to express green fluorescent protein (GFP). A 10-μL volume of virus in tripartite soil (bovine serum albumin, tryptone, and mucin) was combined with 50 μL of disinfectant in suspension for 0.5, 1, 5, or 10 min at 20-25 °C. Neutralized test mixtures were quantified by GFP expression to determine log reduction. Remaining material was passaged on Vero cells to confirm absence of residual infectious virus. Input virus titers of 6.6-8.0 log per assay were completely inactivated by each disinfectant within 1-5 min contact time. The rapid and substantial inactivation of LASV suggests the utility of these microbicides for mitigating spread of infectious virus during Lassa fever outbreaks.
Topics: Animals; Chlorocebus aethiops; Humans; Lassa virus; Lassa Fever; Vero Cells; Anti-Infective Agents; Disinfectants; Green Fluorescent Proteins
PubMed: 37563252
DOI: 10.1038/s41598-023-38954-5 -
Emerging Microbes & Infections Dec 2024Lassa virus (LASV) is a World Health Organization (WHO) priority pathogen that causes high morbidity and mortality. Recently, we showed that a combination of three...
Lassa virus (LASV) is a World Health Organization (WHO) priority pathogen that causes high morbidity and mortality. Recently, we showed that a combination of three broadly neutralizing human monoclonal antibodies known as Arevirumab-3 (8.9F, 12.1F, 37.2D) based on the lineage IV Josiah strain protected 100% of cynomolgus macaques against heterologous challenge with lineage II and III strains of LASV when therapy was initiated beginning at day 8 after challenge. LASV strains from Benin and Togo represent a new lineage VII that are more genetically diverse from lineage IV than strains from lineages II and III. Here, we tested the ability of Arevirumab-3 to protect macaques against a LASV lineage VII Togo isolate when treatment was administered beginning 8 days after exposure. Unexpectedly, only 40% of treated animals survived challenge. In a subsequent study we showed that Arevirumab-3 protected 100% of macaques from lethal challenge when treatment was initiated 7 days after LASV Togo exposure. Based on our transcriptomics data, successful Arevirumab-3 treatment correlated with diminished neutrophil signatures and the predicted development of T cell responses. As the antiviral activity of Arevirumab-3 against LASV Togo was equivalent to lineage II and III strains, the reduced protection in macaques against Togo likely reflects the faster disease course of LASV Togo in macaques than other strains. This data causes concern regarding the ability of heterologous vaccines and treatments to provide cross protection against lineage VII LASV isolates.
Topics: Humans; Animals; Lassa virus; Lassa Fever; Virulence; Macaca fascicularis; Antibodies, Monoclonal
PubMed: 38164768
DOI: 10.1080/22221751.2023.2301061 -
PLoS Pathogens Jul 2023Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral...
Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.
Topics: Arenavirus; Viroporin Proteins; Glycoproteins; Viral Envelope Proteins; Lassa virus; Virus Internalization
PubMed: 37494374
DOI: 10.1371/journal.ppat.1011217 -
Virology Journal Dec 2023Lymphocytic choriomeningitis virus (LCMV) is a human pathogen naturally present in wild rodents. In addition, LCMV is routinely used in immunology research as a model of...
BACKGROUND
Lymphocytic choriomeningitis virus (LCMV) is a human pathogen naturally present in wild rodents. In addition, LCMV is routinely used in immunology research as a model of viral infection in mice. The Armstrong common laboratory strain and the Clone-13 variant induce acute and chronic infections in mice, respectively. The frequent use of this virus in laboratory settings is associated with a risk of human infection for laboratory personnel. In contrast to LCMV Clone-13, few human laboratory infections with LCMV Armstrong have been reported, leading to a poor understanding of symptoms related to infection with this specific LCMV strain.
CASE PRESENTATION
A researcher accidentally infected herself percutaneously with LCMV Armstrong. Symptoms including headaches, dizziness, eye pain and nausea appeared seven days post-exposure and lasted ten days. LCMV-IgM antibodies were detected at 28 days post-infection and IgG seroconversion was observed later. Complete recovery was confirmed three months post exposure.
CONCLUSIONS
Research involving live viruses comes with the risk of infection for research personnel. This case is the first reported accidental human infection with LCMV Armstrong. The symptoms differed from reported infections with LCMV Clone-13, by the absence of fever and vomiting, and presence of leg numbness. This report will therefore help clinicians and public health authorities to recognize the symptoms associated with LCMV Armstrong infections and to offer appropriate counselling to individuals who accidentally expose themselves.
Topics: Animals; Humans; Mice; Antibodies, Viral; Immunoglobulin M; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Mice, Inbred C57BL; Rodentia; Female
PubMed: 38087355
DOI: 10.1186/s12985-023-02258-x -
Journal of the American Chemical Society Dec 2023Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the...
Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs must be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly, and budding and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA, and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z, and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for the disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment, and release in Lassa virus.
Topics: Lassa virus; Ribonucleoproteins; Nucleoproteins; Virus Assembly; RNA, Viral
PubMed: 38104324
DOI: 10.1021/jacs.3c07325 -
Frontiers in Immunology 2024Macrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments...
INTRODUCTION
Macrophage function is determined by microenvironment and origin. Brain and retinal microglia are both derived from yolk sac progenitors, yet their microenvironments differ. Utilizing single-cell RNA sequencing (scRNA-seq) data from mice, we tested the hypothesis that retinal and brain microglia exhibit distinct transcriptional profiles due to their unique microenvironments.
METHODS
Eyes and brains from 2-4 month wildtype mice were combined (20 eyes; 3 brains) to yield one biologically diverse sample per organ. Each tissue was digested into single cell suspensions, enriched for immune cells, and sorted for scRNA-seq. Analysis was performed in Seurat v3 including clustering, integration, and differential expression. Multi-parameter flow cytometry was used for validation of scRNA-seq results. Lymphocytic choriomeningitis virus (LCMV) Clone 13, which produces a systemic, chronic, and neurotropic infection, was used to validate scRNA-seq and flow cytometry results .
RESULTS
Cluster analysis of integrated gene expression data from eye and brain identified 6 microglial clusters. Differential expression analysis revealed that eye microglia were enriched for more pro-inflammatory processes including antigen processing via MHC class I (14.0-fold, and ) and positive regulation of T-cell immunity (8.4-fold) compared to brain microglia. Multi-parameter flow cytometry confirmed that retinal microglia expressed 3.2-fold greater H2-Db and 263.3-fold more H2-Kb than brain microglia. On Day 13 and 29 after LCMV infection, CD8 T-cell density was greater in the retina than the brain.
DISCUSSION
Our data demonstrate that the microenvironment of retina and brain differs, resulting in microglia-specific gene expression changes. Specifically, retinal microglia express greater MHC class I by scRNA-seq and multi-parameter flow cytometry, resulting in a possibly enhanced capability to stimulate CD8 T-cell inflammation during LCMV infection. These results may explain tissue-specific differences between retina and brain during systemic viral infections and CD8 T-cell driven autoimmune disease.
Topics: Animals; Microglia; Mice; Retina; Brain; Mice, Inbred C57BL; Lymphocytic choriomeningitis virus; Histocompatibility Antigens Class I; T-Lymphocytes; Inflammation; Lymphocytic Choriomeningitis; Single-Cell Analysis; CD8-Positive T-Lymphocytes; Transcriptome
PubMed: 38799448
DOI: 10.3389/fimmu.2024.1399989 -
Viruses Jan 2024Metagenomic analysis of and mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant...
Metagenomic analysis of and mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant reads found in mosquitoes were of Phasi Charoen-like virus (PCLV), granulovirus (CfGV), Cell fusing agent virus (CFAV), and Wenzhou sobemo-like virus 4 (WSLV4), whereas WSLV4 and CfGV constituted the highest percentage of reads in viromes. Other reads that were of low percentage included Hubei mosquito virus 2 (HMV2), Porcine astrovirus 4 (PAstV4), and Wild Boar astrovirus (WBAstV). PCLV and CFAV, which were found to be abundant in . viromes were absent in viromes. Among the viromes analyzed, sampled from Pune showed the highest percentage (79.82%) of viral reads, while . mosquitoes sampled from Dibrugarh showed the lowest percentage (3.47%). Shamonda orthobunyavirus (SHAV), African swine fever virus (ASFV), Aroa virus (AROAV), and Ilheus virus (ILHV), having the potential to infect vertebrates, including humans, were also detected in both mosquito species, albeit with low read numbers. Reads of gemykibivirus, avian retrovirus, bacteriophages, herpesviruses, and viruses infecting protozoans, algae, etc., were also detected in the mosquitoes. A high percentage of reads in the mosquito samples belonged to unclassified viruses and warrant further investigation. The data generated in the present work may not only lead to studies to explain the influence of these viruses on the replication and transmission of viruses of clinical importance but also to find applications as biocontrol agents against pathogenic viruses.
Topics: Animals; Swine; Humans; Aedes; African Swine Fever Virus; Virome; India; Bacteriophages; Arenaviridae; Granulovirus
PubMed: 38257809
DOI: 10.3390/v16010109 -
Euro Surveillance : Bulletin Europeen... Oct 2023BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and...
BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and clinical awareness. Therefore, it is important to regularly evaluate laboratory diagnostic capabilities, e.g. by external quality assessments (EQA).AimWe wished to evaluate the performance and diagnostic capability of European expert laboratories to detect orthohantaviruses and lymphocytic choriomeningitis virus (LCMV) and human antibody response towards orthohantaviruses.MethodsWe conducted an EQA in 2021; molecular panels consisted of 12 samples, including different orthohantaviruses (Seoul, Dobrava-Belgrade (DOBV), Puumala (PUUV) and Hantaan orthohantavirus), LCMV and negative controls. Serological panels consisted of six human serum samples reactive to PUUV, DOBV or negative to orthohantaviruses. The EQA was sent to 25 laboratories in 20 countries.ResultsThe accuracy of molecular detection of orthohantaviruses varied (50‒67%, average 62%) among 16 participating laboratories, while LCMV samples were successfully detected in all 11 participating laboratories (91-100%, average 96%). The accuracy of serological diagnosis of acute and past orthohantavirus infections was on average 95% among 20 participating laboratories and 82% in 19 laboratories, respectively. A variety of methods was used, with predominance of in-house assays for molecular tests, and commercial assays for serological ones.ConclusionSerology, the most common tool to diagnose acute orthohantavirus infections, had a high accuracy in this EQA. The molecular detection of orthohantaviruses needs improvement while LCMV detection (performed in fewer laboratories) had 95% accuracy. Further EQAs are recommended to be performed periodically to monitor improvements and challenges in the diagnostics of rodent-borne diseases.
Topics: Humans; Lymphocytic choriomeningitis virus; Orthohantavirus; Europe; Hantavirus Infections; Antibodies, Viral
PubMed: 37796441
DOI: 10.2807/1560-7917.ES.2023.28.40.2300054