Infected Host Cells Research Articles

ANTIBACTERIAL AND ANTIVIRAL ACTIVITIES OF STINGING NETTLE (URTICA DIOICA L.) LEAF EXTRACT ON NOROVIRUS AND CAMPYLOBACTER JEJUNI AS FOODBORNE PATHOGENS

Research background. The cytotoxicity on various vital cell lines and the activity on foodborne pathogens (murine norovirus 1 as a norovirus surrogate and Campylobacter jejuni) of methanolic Urtica dioica L. leaf extract (UDE) were studied. Experimental approach. The cytotoxic concentration of 50% (CC50) was measured by the linearity between UDE concentrations and cell viability. Antibacterial effects on C. jejuni were analyzed by the broth microdilution method with a spectrophotometer. The virucidal and antiviral activities of UDE were determined by the virus titration method on the host cell infectivity and expressed as the tissue cell infective dose of 50% using the method of Spearman–Karber. Results and conclusions. The CC50 of UDE was determined on macrophage as the virus host cell. MIC and MBC of UDE were determined as 5 mg/ml and 20 mg/ml for both C. jejuni isolated from poultry meat and the standard strain. UDE inhibited MNV-1 on three pathways of host cell infectivity at approximately the same 50% inhibitory concentration (1.45-1.87 mg/ml). In conclusion, the present study tried to explain in detail the dose-dependent activity of Urtica dioica L. leaf extract on two important foodborne pathogens causing outbreaks worldwide. The results showed that it might be a safe and alternative food additive and supplement candidate at safe concentrations.

The Role of Spermidine and Its Key Metabolites in Important, Pathogenic Human Viruses and in Parasitic Infections Caused by Plasmodium falciparum and Trypanosoma brucei.

The triamine spermidine is a key metabolite of the polyamine pathway. It plays a crucial role in many infectious diseases caused by viral or parasitic infections. Spermidine and its metabolizing enzymes, i.e., spermidine/spermine-N1-acetyltransferase, spermine oxidase, acetyl polyamine oxidase, and deoxyhypusine synthase, fulfill common functions during infection in parasitic protozoa and viruses which are obligate, intracellular parasites. The competition for this important polyamine between the infected host cell and the pathogen determines the severity of infection in disabling human parasites and pathogenic viruses. Here, we review the impact of spermidine and its metabolites in disease development of the most important, pathogenic human viruses such as SARS-CoV-2, HIV, Ebola, and in the human parasites Plasmodium and Trypanosomes. Moreover, state-of-the-art translational approaches to manipulate spermidine metabolism in the host and the pathogen are discussed to accelerate drug development against these threatful, infectious human diseases.

PcTrim prevents early infection with white spot syndrome virus by inhibiting AP1-induced endocytosis

Viruses have evolved various strategies to achieve early infection by initiating transcription of their own early genes via host transcription factors, such as NF-κb, STAT, and AP1. How the host copes with this immune escape has been a topic of interest. Tripartite motif (TRIM) family proteins with RING-type domains have E3 ubiquitin ligase activity and are known as host restriction factors. Trim has been reported to be associated with phagocytosis and is also believed to be involved in the activation of autophagy. Preventing the virus from entering the host cell may be the most economical way for the host to resist virus infection. The role of TRIM in the early stage of virus infection in host cells remains to be further interpreted. In the current study, a crayfish TRIM with a RING-type domain, designated as PcTrim, was significantly upregulated under white spot syndrome virus (WSSV) infection in the red swamp crayfish (Procambarus clarkii). Recombinant PcTrim significantly inhibited WSSV replication in crayfish. RNAi targeting PcTrim or blocking PcTrim with an antibody promoted WSSV replication in crayfish. Pulldown and co-IP assays showed that PcTrim can interact with the virus protein VP26. PcTrim restricts the expression level of dynamin, which is involved in the regulation of phagocytosis, by inhibiting AP1 entry into the nucleus. AP1-RNAi effectively reduced the expression levels of dynamin and inhibited host cell endocytosis of WSSV in vivo. Our study demonstrated that PcTrim might reduce early WSSV infection by binding to VP26 and then inhibiting AP1 activation, resulting in reduced endocytosis of WSSV in crayfish hemocytes.BjibKXcktbHxsdfgurhU3PVideo

Density estimation of SARS-CoV2 spike proteins using super pixels segmentation technique

The worldwide outbreak of COVID-19 disease was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2). The existence of spike proteins, which allow these viruses to infect host cells, is one of the distinctive biological traits of various prior viruses. As a result, the process by which these viruses infect people is largely dependent on spike proteins. The density of SARS-CoV-2 spike proteins must be estimated to better understand and develop diagnostics and vaccines against the COVID-19 pandemic. CT scans and X-rays have three issues: frosted glass, consolidation, and strange roadway layouts. Each of these issues can be graded separately or together. Although CT scan is sensitive to COVID-19, it is not very specific. Therefore, patients who obtain these results should have more comprehensive clinical and laboratory tests to rule out other probable reasons. This work collected 586 SARS-CoV 2 transmission electron microscopy (TEM) images from open source for density estimation of virus spike proteins through a segmentation approach based on the superpixel technique. As a result, the spike density means of SARS-CoV2 and SARS-CoV were 21,97nm and 22,45nm, respectively. Furthermore, in the future, we aim to include this model in an intelligent system to enhance the accuracy of viral detection and classification. Moreover, we can remotely connect hospitals and public sites to conduct environmental hazard assessments and data collection.

The metastable associations of bacteriophages and Erwinia amylovora.

Bacteriophages are often considered as possible agents of biological control of unwanted bacterial populations in medicine, agriculture and food industry. Although the virulent phages can efficiently kill the infected host cells but at the population level phage attack not always leads to the host population collapse but may result in establishment of a more or less stable co-existence. The mechanism of the long-term stabilization of the mixed phage-host cultures is poorly understood. Here we describe bacteriophages VyarbaL and Hena2, the members of the Molineuxvirinae and the Ounavirinae subfamilies, respectively, that are able to form the pseudolysogenic associations (PA) with their host Erwinia amylovora 1/79Sm on solid media. These PAs were stable through multiple passages. The phenomenon of the PA formation between a bacterial culture and bacteriophages decreases the effectiveness of bacteriophage-mediated biological control agents based on lytic bacteriophages.

Three-Dimensional Chromatin Structure of the EBV Genome: A Crucial Factor in Viral Infection.

Epstein-Barr Virus (EBV) is a human gamma-herpesvirus that is widespread worldwide. To this day, about 200,000 cancer cases per year are attributed to EBV infection. EBV is capable of infecting both B cells and epithelial cells. Upon entry, viral DNA reaches the nucleus and undergoes a process of circularization and chromatinization and establishes a latent lifelong infection in host cells. There are different types of latency all characterized by different expressions of latent viral genes correlated with a different three-dimensional architecture of the viral genome. There are multiple factors involved in the regulation and maintenance of this three-dimensional organization, such as CTCF, PARP1, MYC and Nuclear Lamina, emphasizing its central role in latency maintenance.

In silico structural and functional characterization of hypothetical proteins from Monkeypox virus

BackgroundMonkeypox virus is a small, double-stranded DNA virus that causes a zoonotic disease called Monkeypox. The disease has spread from Central and West Africa to Europe and North America and created havoc in some countries all around the world. The complete genome of the Monkeypox virus Zaire-96-I-16 has been sequenced. The viral strain contains 191 protein-coding genes with 30 hypothetical proteins whose structure and function are still unknown. Hence, it is imperative to functionally and structurally annotate the hypothetical proteins to get a clear understanding of novel drug and vaccine targets. The purpose of the study was to characterize the 30 hypothetical proteins through the determination of physicochemical properties, subcellular characterization, function prediction, functional domain prediction, structure prediction, structure validation, structural analysis, and ligand binding sites using Bioinformatics tools. ResultsThe structural and functional analysis of 30 hypothetical proteins was carried out in this research. Out of these, 3 hypothetical functions (Q8V547, Q8V4S4, Q8V4Q4) could be assigned a structure and function confidently. Q8V547 protein in Monkeypox virus Zaire-96-I-16 is predicted as an apoptosis regulator which promotes viral replication in the infected host cell. Q8V4S4 is predicted as a nuclease responsible for viral evasion in the host. The function of Q8V4Q4 is to prevent host NF-kappa-B activation in response to pro-inflammatory cytokines like TNF alpha or interleukin 1 beta. ConclusionsOut of the 30 hypothetical proteins of Monkeypox virus Zaire-96-I-16, 3 were annotated using various bioinformatics tools. These proteins function as apoptosis regulators, nuclease, and inhibitors of NF-Kappa-B activator. The functional and structural annotation of the proteins can be used to perform a docking with potential leads to discover novel drugs and vaccines against the Monkeypox. In vivo research can be carried out to identify the complete potential of the annotated proteins.

Identification and Comparison of the Sialic Acid-Binding Domain Characteristics of Avian Coronavirus Infectious Bronchitis Virus Spike Protein.

Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.

The Periplasmic Tail-Specific Protease, Tsp, Is Essential for Secondary Differentiation in Chlamydia trachomatis.

The obligate intracellular human pathogen Chlamydia trachomatis (Ctr) undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms: the elementary body (EB) and the reticulate body (RB). The EB is the smaller, infectious, nondividing form which initiates infection of a susceptible host cell, whereas the RB is the larger, non-infectious form which replicates within a membrane-bound vesicle called an inclusion. The mechanism(s) which drives differentiation between these developmental forms is poorly understood. Bulk protein turnover is likely required for chlamydial differentiation given the significant differences in the protein repertoires and functions of the EB and RB. We hypothesize that periplasmic protein turnover is also critical for the reorganization of an RB into an EB, referred to as secondary differentiation. Ct441 is a periplasmic protease ortholog of tail-specific proteases (i.e., Tsp, Prc) and is expressed in Ctr during secondary differentiation. We investigated the effect of altering Tsp expression on developmental cycle progression. Through assessment of bacterial morphology and infectious progeny production, we found that both overexpression and CRISPR interference/dCas9 (CRISPRi)-mediated knockdown of Tsp negatively impacted chlamydial development through different mechanisms. We also confirmed that catalytic activity is required for the negative effect of overexpression and confirmed the effect of the mutation in in vitro assays. Electron microscopic assessments during knockdown experiments revealed a defect in EB morphology, directly linking Tsp function to secondary differentiation. These data implicate Ct441/Tsp as a critical factor in secondary differentiation. IMPORTANCE The human pathogen Chlamydia trachomatis is the leading cause of preventable infectious blindness and bacterial sexually transmitted infections worldwide. This pathogen has a unique developmental cycle that alternates between distinct forms. However, the key processes of chlamydial development remain obscure. Uncovering the mechanisms of differentiation between its metabolically and functionally distinct developmental forms may foster the discovery of novel Chlamydia-specific therapeutics and limit development of resistant bacterial populations derived from the clinical use of broad-spectrum antibiotics. In this study, we investigate chlamydial tail-specific protease (Tsp) and its function in chlamydial growth and development. Our work implicates Tsp as essential to chlamydial developmental cycle progression and indicates that Tsp is a potential drug target for Chlamydia infections.

Discovery of novel papain-like protease inhibitors for potential treatment of COVID-19

The recent emergence of different SARS-CoV-2 variants creates an urgent need to develop more effective therapeutic agents to prevent COVID-19 outbreaks. Among SARS-CoV-2 essential proteases is papain-like protease (SARS-CoV-2 PLpro), which plays multiple roles in regulating SARS-CoV-2 viral spread and innate immunity such as deubiquitinating and deISG15ylating (interferon-induced gene 15) activities. Many studies are currently focused on targeting this protease to tackle SARS-CoV-2 infection. In this context, we performed a phenotypic screening using an in-house pilot compounds collection possessing a diverse skeleta against SARS-CoV-2 PLpro. This screen identified SIMR3030 as a potent inhibitor of SARS-CoV-2. SIMR3030 has been shown to exhibit deubiquitinating activity and inhibition of SARS-CoV-2 specific gene expression (ORF1b and Spike) in infected host cells and possessing virucidal activity. Moreover, SIMR3030 was demonstrated to inhibit the expression of inflammatory markers, including IFN-α, IL-6, and OAS1, which are reported to mediate the development of cytokine storms and aggressive immune responses. In vitro absorption, distribution, metabolism, and excretion (ADME) assessment of the drug-likeness properties of SIMR3030 demonstrated good microsomal stability in liver microsomes. Furthermore, SIMR3030 demonstrated very low potency as an inhibitor of CYP450, CYP3A4, CYP2D6 and CYP2C9 which rules out any potential drug-drug interactions. In addition, SIMR3030 showed moderate permeability in Caco2-cells. Critically, SIMR3030 has maintained a high in vivo safety profile at different concentrations. Molecular modeling studies of SIMR3030 in the active sites of SARS-CoV-2 and MERS-CoV PLpro were performed to shed light on the binding modes of this inhibitor. This study demonstrates that SIMR3030 is a potent inhibitor of SARS-CoV-2 PLpro that forms the foundation for developing new drugs to tackle the COVID-19 pandemic and may pave the way for the development of novel therapeutics for a possible future outbreak of new SARS-CoV-2 variants or other Coronavirus species.

Oncolytic Rodent Protoparvoviruses Evade a TLR- and RLR-Independent Antiviral Response in Transformed Cells.

The oncolytic rodent protoparvoviruses (PVs) minute virus of mice (MVMp) and H-1 parvovirus (H-1PV) are promising cancer viro-immunotherapy candidates capable of both exhibiting direct oncolytic activities and inducing anticancer immune responses (AIRs). Type-I interferon (IFN) production is instrumental for the activation of an efficient AIR. The present study aims at characterizing the molecular mechanisms underlying PV modulation of IFN induction in host cells. MVMp and H-1PV triggered IFN production in semi-permissive normal mouse embryonic fibroblasts (MEFs) and human peripheral blood mononuclear cells (PBMCs), but not in permissive transformed/tumor cells. IFN production triggered by MVMp in primary MEFs required PV replication and was independent of the pattern recognition receptors (PRRs) Toll-like (TLR) and RIG-like (RLR) receptors. PV infection of (semi-)permissive cells, whether transformed or not, led to nuclear translocation of the transcription factors NFĸB and IRF3, hallmarks of PRR signaling activation. Further evidence showed that PV replication in (semi-)permissive cells resulted in nuclear accumulation of dsRNAs capable of activating mitochondrial antiviral signaling (MAVS)-dependent cytosolic RLR signaling upon transfection into naïve cells. This PRR signaling was aborted in PV-infected neoplastic cells, in which no IFN production was detected. Furthermore, MEF immortalization was sufficient to strongly reduce PV-induced IFN production. Pre-infection of transformed/tumor but not of normal cells with MVMp or H-1PV prevented IFN production by classical RLR ligands. Altogether, our data indicate that natural rodent PVs regulate the antiviral innate immune machinery in infected host cells through a complex mechanism. In particular, while rodent PV replication in (semi-)permissive cells engages a TLR-/RLR-independent PRR pathway, in transformed/tumor cells this process is arrested prior to IFN production. This virus-triggered evasion mechanism involves a viral factor(s), which exert(s) an inhibitory action on IFN production, particularly in transformed/tumor cells. These findings pave the way for the development of second-generation PVs that are defective in this evasion mechanism and therefore endowed with increased immunostimulatory potential through their ability to induce IFN production in infected tumor cells.

Gender trend of monkeypox virus infection

ABSTRACT Introduction An outbreak of monkeypox (mpox) in 2022 has been declared as a ‘public health emergency of international concern’ by the World Health Organization (WHO). There are many reports about the cases of male-to-male transmission of the recent mpox virus (MPXV). However, the mechanism and trend of male infection are unclear. We analyzed public data to test whether men are vulnerable to mpox by gender effect. Areas covered Public data of previously and recently reported cohort cases, including gender information of MPXV-infected patients, from PubMed, Google Scholar, and EBSCO databases were collected and analyzed. Network analysis was used to explore the potent intersections between male hormone receptor, androgen receptor (AR) signaling, and mpox-related and -infected host cell response genes. Furthermore, gene ontology enrichment and KEGG genomic signaling pathways were analyzed using intersection genes. Expert opinion MPXV infections among the male population are more frequent than the female population using multiple cohort public data analysis. AR signaling-related gene list against mpox host cell response gene list data of two sets showed that the most intersection genes are related to human immunodeficiency virus (HIV) infection, inflammation, and transcription. AR signaling may be essential to the infection and might be a potent target in anti-mpox infective therapy.

Activation of the Plasmodium Egress Effector Subtilisin-Like Protease 1 Is Mediated by Plasmepsin X Destruction of the Prodomain.

Following each round of replication, daughter merozoites of the malaria parasite Plasmodium falciparum escape (egress) from the infected host red blood cell (RBC) by rupturing the parasitophorous vacuole membrane (PVM) and the RBC membrane (RBCM). A proteolytic cascade orchestrated by a parasite serine protease, subtilisin-like protease 1 (SUB1), regulates the membrane breakdown. SUB1 activation involves primary autoprocessing of the 82-kDa zymogen to a 54-kDa (p54) intermediate that remains bound to its inhibitory propiece (p31) postcleavage. A second processing step converts p54 to the terminal 47-kDa (p47) form of SUB1. Although the aspartic protease plasmepsin X (PM X) has been implicated in the activation of SUB1, the mechanism remains unknown. Here, we show that upon knockdown of PM X, the inhibitory p31-p54 complex of SUB1 accumulates in the parasites. Using recombinant PM X and SUB1, we show that PM X can directly cleave both p31 and p54. We have mapped the cleavage sites on recombinant p31. Furthermore, we demonstrate that the conversion of p54 to p47 can be effected by cleavage at either SUB1 or PM X cleavage sites that are adjacent to one another. Importantly, once the p31 is removed, p54 is fully functional inside the parasites, suggesting that the conversion to p47 is dispensable for SUB1 activity. Relief of propiece inhibition via a heterologous protease is a novel mechanism for subtilisin activation. IMPORTANCE Malaria parasites replicate inside a parasitophorous vacuole within the host red blood cells. The exit of mature progeny from the infected host cells is essential for further dissemination. Parasite exit is a highly regulated, explosive process that involves membrane breakdown. To do this, the parasite utilizes a serine protease called SUB1 that proteolytically activates various effector proteins. SUB1 activity is dependent on an upstream protease called PM X, although the mechanism was unknown. Here, we describe the molecular basis for PM X-mediated SUB1 activation. PM X proteolytically degrades the inhibitory segment of SUB1, thereby activating it. The involvement of a heterologous protease is a novel mechanism for subtilisin activation.

BopN is a Gatekeeper of the Bordetella Type III Secretion System.

The classical Bordetella species infect the respiratory tract of mammals. While B. bronchiseptica causes rather chronic respiratory infections in a variety of mammals, the human-adapted species B. pertussis and B. parapertussisHU cause an acute respiratory disease known as whooping cough or pertussis. The virulence factors include a type III secretion system (T3SS) that translocates effectors BteA and BopN into host cells. However, the regulatory mechanisms underlying the secretion and translocation activity of T3SS in bordetellae are largely unknown. We have solved the crystal structure of BopN of B. pertussis and show that it is similar to the structures of gatekeepers that control access to the T3SS channel from the bacterial cytoplasm. We further found that BopN accumulates at the cell periphery at physiological concentrations of calcium ions (2 mM) that inhibit the secretion of BteA and BopN. Deletion of the bopN gene in B. bronchiseptica increased secretion of the BteA effector into calcium-rich medium but had no effect on secretion of the T3SS translocon components BopD and BopB. Moreover, the ΔbopN mutant secreted approximately 10-fold higher amounts of BteA into the medium of infected cells than the wild-type bacteria, but it translocated lower amounts of BteA into the host cell cytoplasm. These data demonstrate that BopN is a Bordetella T3SS gatekeeper required for regulated and targeted translocation of the BteA effector through the T3SS injectisome into host cells. IMPORTANCE The T3SS is utilized by many Gram-negative bacteria to deliver effector proteins from bacterial cytosol directly into infected host cell cytoplasm in a regulated and targeted manner. Pathogenic bordetellae use the T3SS to inject the BteA and BopN proteins into infected cells and upregulate the production of the anti-inflammatory cytokine interleukin-10 (IL-10) to evade host immunity. Previous studies proposed that BopN acted as an effector in host cells. In this study, we report that BopN is a T3SS gatekeeper that regulates the secretion and translocation activity of Bordetella T3SS.

Epstein-Barr Virus Envelope Glycoprotein gp110 Inhibits IKKi-Mediated Activation of NF-κB and Promotes the Degradation of β-Catenin.

Epstein-Barr virus (EBV) infects host cells and establishes a latent infection that requires evasion of host innate immunity. A variety of EBV-encoded proteins that manipulate the innate immune system have been reported, but whether other EBV proteins participate in this process is unclear. EBV-encoded envelope glycoprotein gp110 is a late protein involved in virus entry into target cells and enhancement of infectivity. Here, we reported that gp110 inhibits RIG-I-like receptor pathway-mediated promoter activity of interferon-β (IFN-β) as well as the transcription of downstream antiviral genes to promote viral proliferation. Mechanistically, gp110 interacts with the inhibitor of NF-κB kinase (IKKi) and restrains its K63-linked polyubiquitination, leading to attenuation of IKKi-mediated activation of NF-κB and repression of the phosphorylation and nuclear translocation of p65. Additionally, gp110 interacts with an important regulator of the Wnt signaling pathway, β-catenin, and induces its K48-linked polyubiquitination degradation via the proteasome system, resulting in the suppression of β-catenin-mediated IFN-β production. Taken together, these results suggest that gp110 is a negative regulator of antiviral immunity, revealing a novel mechanism of EBV immune evasion during lytic infection. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous pathogen that infects almost all human beings, and the persistence of EBV in the host is largely due to immune escape mediated by its encoded products. Thus, elucidation of EBV's immune escape mechanisms will provide a new direction for the design of novel antiviral strategies and vaccine development. Here, we report that EBV-encoded gp110 serves as a novel viral immune evasion factor, which inhibits RIG-I-like receptor pathway-mediated interferon-β (IFN-β) production. Furthermore, we found that gp110 targeted two key proteins, inhibitor of NF-κB kinase (IKKi) and β-catenin, which mediate antiviral activity and the production of IFN-β. gp110 inhibited K63-linked polyubiquitination of IKKi and induced β-catenin degradation via the proteasome, resulting in decreased IFN-β production. In summary, our data provide new insights into the EBV-mediated immune evasion surveillance strategy.

Development of an inhalable antiviral powder formulation against respiratory syncytial virus

Respiratory viruses including the respiratory syncytial virus (RSV) aggravate the global burden of virus-inflicted morbidity and mortality. Entry inhibitors are a promising class of antiviral drugs for combating these viruses, as they can prevent infection at the site of viral entry, i.e., the respiratory tract. Here we used a broad-spectrum entry inhibitor, composed of a β-cyclodextrin backbone, functionalized with 11-mercapto-1-undecanesulfonate (CD-MUS) that is capable of neutralizing a variety of viruses that employ heparan sulfate proteoglycans (HSPG) to infect host cells. CD-MUS inactivates viral particles irreversibly by binding to viral attachment proteins through a multivalent binding mechanism. In the present study, we show that CD-MUS is well tolerated when administered to the respiratory tract of mice. Based on this, we developed an inhalable spray-dried powder formulation that fits the size requirements for lung deposition and disperses well upon use with the Cyclops dry powder inhaler (DPI). Using an in vitro dose-response assay, we show that the compound retained its activity against RSV after the spray drying process. Our study sets the stage for further in vivo studies, exploring the efficacy of pulmonary administered CD-MUS in animal models of RSV infection.

Analysis of Morphological Changes in the Nucleus and Vacuoles of Acanthamoeba castellanii following Giant Virus Infection.

Acanthamoeba castellanii medusavirus is a member of the phylum Nucleocytoviricota, also known as giant viruses, and has a unique strategy of infecting Acanthamoeba castellanii and replicating viral genes in the host nucleus. Here, we show time series changes in the intracellular morphology, including the nucleus, of host cells infected with four types of giant viruses, including medusavirus, using time-lapse phase-contrast microscopy and image analysis. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) to use multiple microscopic images with different focus positions to allow a more detailed analysis of their intracellular structures. Image analysis using PKA3 revealed that as medusavirus infection progressed, the host nucleus increased in size and the number of vacuoles decreased. In addition, infected host cells are known to become smaller and rounder at later stages of infection, but here they were found to be larger than uninfected cells at earlier stages. These results suggested that the propagation mechanism of medusavirus includes the formation of empty virus particles in the host cytoplasm, packaging of the viral genome replicated in the host nucleus, and then the release of viral particles. IMPORTANCE In this study, we quantitatively revealed how long the increase in host cell size or the increase in host nucleus size occurs after infection with giant viruses, especially medusavirus. To understand the underlying mechanism, we performed image analysis and determined that the host cell size increased at approximately 6 h postinfection (hpi) and the host nucleus enlarged at approximately 22 hpi, pointing to the importance of biochemical experiments. In addition, we showed that the intracellular structures could be quantitatively analyzed using multiple phase-contrast microscopy images with different focus positions at the same time point. Hence, morphological analyses of intracellular structures using phase-contrast microscopy, which have wide applications in live-cell observations, may be useful in studying various organisms that infect or are symbiotic with A. castellanii.

Assessment of the Macrophage Scavenger Receptor CD163 in Mediating Glaesserella parasuis Infection of Host Cells.

The macrophage CD163 surface glycoprotein is a member of the SRCR family class B, which has been identified as the key trigger in host-pathogen interactions, but its specific roles in sensing Glaesserella parasuis (G. parasuis) infection are largely unknown. Here, we investigated porcine CD163 in mediating the adhesion and immune response of G. parasuis using in vitro host-bacteria interaction models. CD163-overexpressing Chinese hamster ovary K1 cells (CHO-K1) showed obvious subcellular localization in the cytoplasm, especially in the cytomembrane. Although detection using scanning electron microscopy (SEM) confirmed the bacterial adhesion, there was no significant difference in the adhesion of G. parasuis to CHO-K1 cells between the presence and absence of CD163. In addition, similar results were observed in 3D4/21 cells. Meanwhile, bindings of G. parasuis to nine synthetic peptides, the bacterial binding motifs within SRCR domains of CD163, were weak based on a solid-phase adhesion assay and agglutination assay. Moreover, CD163 had no effect on the expression of G. parasuis-induced inflammatory cytokines (IL-6, INF-γ, IL-10, IL-4 and TGF-β) in CHO-K1 cells. In conclusion, these findings indicate that porcine CD163 plays a minor role in sensing G. parasuis infection.

Actin based motility unveiled: How chemical energy is converted into motion

Actin-based motility is a complex process in which the actin-polymerization is the primary force-generating motor machinery. It can produce protrusive forces through actin filaments polymerization and cross-link during lamellipodia protrusion in migrating cells and it is responsible for the intracellular motion of certain pathogens in infected host cells. We propose a multi-physics model for actin-based motility, stemming from continuity equations that account for the actin chemical kinetics. Thermodynamic restrictions are identified, moving from the multiplicative decomposition of the deformation gradient into chemical and elastic parts. Constitutive theory and chemical kinetics are prescribed to finally write governing equations for the multi-physics problem. The field equations are solved numerically with the finite element method. As a proof of concept, a one-dimensional model for actin-based motility of bacteria pathogens is studied.

Manipulation of the Cellular Membrane-Cytoskeleton Network for RNA Virus Replication and Movement in Plants.

Viruses infect all cellular life forms and cause various diseases and significant economic losses worldwide. The majority of viruses are positive-sense RNA viruses. A common feature of infection by diverse RNA viruses is to induce the formation of altered membrane structures in infected host cells. Indeed, upon entry into host cells, plant-infecting RNA viruses target preferred organelles of the cellular endomembrane system and remodel organellar membranes to form organelle-like structures for virus genome replication, termed as the viral replication organelle (VRO) or the viral replication complex (VRC). Different viruses may recruit different host factors for membrane modifications. These membrane-enclosed virus-induced replication factories provide an optimum, protective microenvironment to concentrate viral and host components for robust viral replication. Although different viruses prefer specific organelles to build VROs, at least some of them have the ability to exploit alternative organellar membranes for replication. Besides being responsible for viral replication, VROs of some viruses can be mobile to reach plasmodesmata (PD) via the endomembrane system, as well as the cytoskeleton machinery. Viral movement protein (MP) and/or MP-associated viral movement complexes also exploit the endomembrane-cytoskeleton network for trafficking to PD where progeny viruses pass through the cell-wall barrier to enter neighboring cells.