Equine herpesvirus-1 (EHV-1) infections cause significant economic losses for equine industries worldwide as a result of abortion, respiratory illness, and neurologic disease in all breeds of horses. The occurrence of abortions caused by EHV-1 has repeatedly been confirmed in Poland, but neurological manifestations of the infection have not been described yet. Also it is unknown how the infection of neurons with non-neuropathogenic strains is regulated. To further understand the virus-neuron interaction we studied two strains of EHV-1 in murine primary neuron cell cultures. Both strains were isolated from aborted fetuses: Rac-H, a reference strain isolated by Woyciechowska in 1959 (Woyciechowska 1960) and Jan-E isolated by Bańbura et al. (Bańbura et al. 2000). Upon infection of primary murine neuronal cell cultures with Jan-E or Rac-H strains, a cytopathic effect was observed, manifested by a changed morphology and disintegration of the cell monolayer. Positive results of immunofluorescence, nPCR and real-time PCR tests indicated high virus concentration in neurons, meaning that both EHV-1 strains were likely to replicate in mouse neurons in vitro without the need for adaptation. Moreover, we demonstrated that some neurons may survive (limited) virus replication during primary infection, and these neurons (eight weeks p.i.) harbour EHV-1 and were still able to transmit infection to other cells.
Mitochondrial movement and distribution throughout the cytoplasm is crucial for maintaining cell homeostasis. Mitochondria are dynamic organelles but can be functionally disrupted during infection. Here, we show that the ubiquitous human pathogens HHV-1 and HHV-2 induce changes in the mitochondrial morphology and distribution in the early and late phases of productive infection in human keratinocytes (HaCaT cells). We observed a decrease in the mitochondrial potential at 2 h postinfection and a decrease in cell vitality at 24 h postinfection. Moreover, we found that mitochondria migrated to the perinuclear area, where HHV-1 and HHV-2 antigens were also observed, mainly in the early stages of infection. Positive results of real-time PCR showed a high level of HHV-1 and HHV-2 DNA in HaCaT cells and culture medium. Our data demonstrate that HHV-1 and HHV-2 cause mitochondrial dysfunction in human keratinocytes.
Herpes simplex virus type 1 (HSV-1) has the ability to replicate in neurons and glial cells and to produce encephalitis leading to neurodegeneration. Accumulated evidence suggests that nitric oxide (NO) is a key molecule in the pathogenesis of neurotropic virus infections. NO can exert both cytoprotective as well as cytotoxic effects in the central nervous system (CNS) depending on its concentration, time course exposure, and site of action. In this study, we used an in vitro model of HSV-1-infected primary neuronal and mixed glial cultures as well as an intranasal model of HSV-1 in BALB/c mice to elucidate the role of NO and nonapoptotic Fas signalling in neuroinflammation and neurodegeneration. We found that low, nontoxic concentration of NO decreased HSV-1 replication in neuronal cultures together with production of IFN-alpha and proinflammatory chemokines. However, in HSV-1-infected glial cultures, low concentrations of NO supported virus replication and production of IFN-alpha and proinflammatory chemokines. HSV-1-infected microglia downregulated Fas expression and upregulated its ligand, FasL. Fas signalling led to production of proinflammatory cytokines and chemokines as well as induced iNOS in uninfected bystander glial cells. On the contrary, NO reduced production of IFN-alpha and CXCL10 through nonapoptotic Fas signalling in HSV-1-infected neuronal cultures. Here, we also observed colocalization of NO production with the accumulation of β-amyloid peptide in HSV-1-infected neurons both in vitro and in vivo. Low levels of the NO donor increased accumulation of β-amyloid in uninfected primary neuronal cultures, while the NO inhibitor decreased its accumulation in HSV-1-infected neuronal cultures. This study shows for the first time the existence of a link between NO and Fas signalling during HSV-1-induced neuroinflammation and neurodegeneration.
Ectromelia virus (ECTV) belongs to the Orthopoxvirus genus of the Poxviridae family and is a natural pathogen of mice. Certain strains of mice are highly susceptible to ECTV infection and develop mousepox, a lethal disease similar to smallpox of humans caused by variola virus. Currently, the mousepox model is one of the available small animal models for investigating pathogenesis of generalized viral infections. Resistance and susceptibility to ECTV infection in mice are controlled by many genetic factors and are associated with multiple mechanisms of immune response, including preferential polarization of T helper (Th) immune response toward Th1 (protective) or Th2 (non-protective) profile. We hypothesized that viral-induced inhibitory effects on immune properties of conventional dendritic cells (cDCs) are more pronounced in ECTV-susceptible than in resistant mouse strains. To this extent, we confronted the cDCs from resistant (C57BL/6) and susceptible (BALB/c) mice with ECTV, regarding their reactivity and potential to drive T cell responses following infection. Our results showed that in vitro infection of granulocyte-macrophage colony-stimulating factor-derived bone marrow cells (GM-BM—comprised of cDCs and macrophages) from C57BL/6 and BALB/c mice similarly down-regulated multiple genes engaged in DC innate and adaptive immune functions, including antigen uptake, processing and presentation, chemokines and cytokines synthesis, and signal transduction. On the contrary, ECTV infection up-regulated Il10 in GM-BM derived from both strains of mice. Moreover, ECTV similarly inhibited surface expression of major histocompatibility complex and costimulatory molecules on GM-BM, explaining the inability of the cells to attain full maturation after Toll-like receptor (TLR)4 agonist treatment. Additionally, cells from both strains of mice failed to produce cytokines and chemokines engaged in T cell priming and Th1/Th2 polarization after TLR4 stimulation. These data strongly suggest that in vitro modulation of GM-BM innate and adaptive immune functions by ECTV occurs irrespective of whether the mouse strain is susceptible or resistant to infection. Moreover, ECTV limits the GM-BM (including cDCs) capacity to stimulate protective Th1 immune response. We cannot exclude that this may be an important factor in the generation of non-protective Th2 immune response in susceptible BALB/c mice in vivo.
Human herpesvirus types 1 and 2 (HHV-1 and HHV-2) are neurotropic viruses which remain latent for life and reactivate to cause recurrent infections. HHV-1 has been found to be involved in accumulation of β-amyloid, hyperphosphorylation of tau proteins, and inflammation in the brain, which can later result in neuronal dysfunction and neurodegeneration. The relationship between HHV-2 and events associated with neurodegeneration has not been extensively studied. Neurons, more than any other cell type, depend on mitochondrial trafficking for their survival, and many types of mitochondrial abnormalities have been described in the etiology of neurodegenerative diseases. Therefore, in this study, we concentrated on mitochondrial dysfunction associated with HHV-1 and HHV-2 infection of primary murine neurons in vitro. We showed that starting from the first stages of HHV-1 and HHV-2 infection, an interaction of viral particles with the mitochondrial network occurs. Both HHV-1 and HHV-2 infection affected mitochondrial function at multiple levels, including upregulation of mitochondrial fission, decrease of the mitochondrial membrane potential, and increase of ROS level. The changes observed in the organization of the mitochondrial network and physiology of productively infected neurons provide appropriate conditions for HHV-1 and HHV-2 replication and are required for effective viral spread.
Equid herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae, is a neurotropic virus, that causes latent infections in the nervous system of the natural host. All alphaherpesviruses have developed sophisticated strategies to interfere with the host cell apoptotic mechanisms, but the ability of EHV-1 to induce apoptosis in neurons has not been determined yet. In this study, apoptotic and necrotic changes in cultured murine neurons were methods identifying key stages of apoptosis. Th ese methods have demonstrated characteristic apoptosis features, like DNA fragmentation, chromatin condensation, membrane blebbing and cell shrinkage in the infected cells. It seems likely that apoptosis was the predominant way of cell death in EHV-1-infected murine neurons. However, we showed also that during acute EHV-1 infection the majority of infected neurons remained unchanged and survived for more than eight weeks in culture, suggesting some protective mechanisms induced by the virus. Furthermore, it was shown that infection of neurons with EHV-1 has no signifi cant infl uence on the level of the caspase 3, 7, and 8. We speculate that the control of apoptosis may be the key mechanism regulating the balance between productive and latent infection at the site of virus persistence. Abbreviations: BHV-1 = bovine herpesvirus 1; EHV-1 = equid herpesvirus 1; HSV-1 = herpes simplex virus 1; HHV-1 = human herpesvirus 1; HHV-2 = human herpesvirus 2; Jan-E = EHV-1 fi eld strain; p.i. = post infection; PI = propidium iodide; PRV = pseudorabies virus; Rac-H = EHV-1 reference strain; TUNEL = Terminal deoxynucleotide transferase dUTP nick end labeling; VZV = varicella zoster virus
Background Polyphenols possess antioxidant, anti-inflammatory and antimicrobial properties and have been used in the treatment of skin wounds and burns. We previously showed that tannic acid-modified AgNPs sized >26 nm promote wound healing, while tannic acid-modified AgNPs sized 13 nm can elicit strong local inflammatory response. In this study, we tested bimetallic Au@AgNPs sized 30 nm modified with selected flavonoid and non-flavonoid compounds for wound healing applications. Methods Bimetallic Au@AgNPs were obtained by growing an Ag layer on AuNPs and further modified with selected polyphenols. After toxicity tests and in vitro scratch assay in HaCaT cells, modified lymph node assay as well as the mouse splint wound model were further used to access the wound healing potential of selected non-toxic modifications. Results Tannic acid, gallic acid, polydatin, resveratrol, catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate and procyanidin B2 used to modify Au@AgNPs exhibited good toxicological profiles in HaCaT cells. Au@AgNPs modified with 15 μM tannic acid, 200 μM resveratrol, 200 μM epicatechin gallate, 1000 μM gallic acid and 200 μM procyanidin B2 induced wound healing in vivo and did not lead to the local irritation or inflammation. Tannic acid-modified Au@AgNPs induced epithelial-to-mesenchymal transition (EMT) – like re-epithelialization, while other polyphenol modifications of Au@AgNPs acted through proliferation and wound closure. Conclusion Bimetallic Au@AgNPs can be used as a basis for modification with selected polyphenols for topical uses. In addition, we have demonstrated that particular polyphenols used to modify bimetallic nanoparticles may show different effects upon different stages of wound healing.
The xCELLigence system for real-time and label-free analysis of neuronal and dermal cell response to Equine Herpesvirus type 1 infection Real-time cell electronic sensing (RT-CES) based on impedance measurements is an emerging technology for analyzing the status of cells in vitro. It allows label-free, real time monitoring of the biological status of cells. The present study was designed to assess dynamic data on the cell processes during equine herpesvirus type 1 (EHV-1) infection of ED (equine dermal) cells and primary murine neuronal cell culture. We have demonstrated that the xCELLigence system with dynamic monitoring can be used as a rapid diagnostic tool both to analyze cellular behavior and to investigate the effect of viral infection.
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