Human Adenovirus Entry and Early Events during Infection of Primary Murine Neurons: Immunofluorescence Studies In Vitro.

Human adenovirus (HAdV) is a common pathogen, which can lead to various clinical symptoms and-in some cases-central nervous system (CNS) dysfunctions, such as encephalitis and meningitis. Although the initial events of virus entry have already been identified in various cell types, the mechanism of neuronal uptake of adenoviruses is relatively little understood. The aim of this study was to investigate early events during adenoviral infection, in particular to determine the connection between cellular coxsackievirus and adenovirus receptor (CAR), clathrin, caveolin, and early endosomal proteins (EEA1 and Rab5) with the entry of HAdVs into primary murine neurons in vitro. An immunofluorescence assay and confocal microscopy analysis were carried out to determine HAdV4, 5, and 7 correlation with CAR, clathrin, caveolin, and early endosomal proteins in neurons. The quantification of Pearson's coefficient between CAR and HAdVs indicated that the HAdV4 and HAdV5 types correlated with CAR and that the correlation was more substantial for HAdV5. Inhibition of clathrin-mediated endocytosis using chlorpromazine limited the infection with HAdV, whereas inhibition of caveolin-mediated endocytosis did not affect virus entry. Thus, the entry of tested HAdV types into neurons was most likely associated with clathrin but not caveolin. It was also demonstrated that HAdVs correlate with the Rab proteins (EEA1, Rab5) present in early vesicles, and the observed differences in the manner of correlation depended on the serotype of the virus. With our research, we strove to expand knowledge regarding the mechanism of HAdV entry into neurons, which may be beneficial for developing potential therapeutics in the future.

Neurons cytoskeletal architecture remodeling during the replication cycle of mouse coronavirus MHV-JHM: a morphological in vitro study.

Nowadays, the population is still struggling with a post-COVID19 syndrome known as long COVID, including a broad spectrum of neurological problems. There is an urgent need for a better understanding and exploration of the mechanisms of coronavirus neurotropism. For this purpose, the neurotropic strain of mouse hepatitis virus (MHV-JHM) originating from the beta-coronavirus genus, the same as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been used. The role of the cytoskeleton during virus replication in neurons in vitro was determined to understand the mechanisms of MHV-JHM neuroinfection. We have described for the first time the changes of actin filaments during MHV-JHM infection. We also observed productive replication of MHV-JHM in neurons during 168 h p.i. and syncytial cytopathic effect. We discovered that the MHV-JHM strain modulated neuronal cytoskeleton during infection, which were manifested by: (i) condensation of actin filaments in the cortical layer of the cytoplasm, (ii) formation of microtubule cisternae structures containing viral antigen targeting viral replication site (iii) formation of tunneling nanotubes used by MHV-JHM for intercellular transport. Additionally, we demonstrated that the use of cytoskeletal inhibitors have reduced virus replication in neurons, especially noscapine and nocodazole, the microtubule shortening factors.

Epigallocatechin Gallate-Modified Silver Nanoparticles Show Antiviral Activity against Herpes Simplex Type 1 and 2.

(1) Background: Epigallocatechin gallate (EGCG) has been recognized as a flavonoid showing antiviral activity against various types of DNA and RNA viruses. In this work, we tested if EGCG-modified silver nanoparticles (EGCG-AgNPs) can become novel microbicides with additional adjuvant properties to treat herpes infections. (2) Methods: The anti-HSV and cytotoxic activities of EGCG-AgNPs were tested in human HaCaT and VK-2-E6/E7 keratinocytes. HSV-1/2 titers and immune responses after treatment with EGCG-AgNPs were tested in murine models of intranasal HSV-1 infection and genital HSV-2 infection. (3) Results: EGCG-AgNPs inhibited attachment and entry of HSV-1 and HSV-2 in human HaCaT and VK-2-E6/E7 keratinocytes much better than EGCG at the same concentration. Infected mice treated intranasally (HSV-1) or intravaginally (HSV-2) with EGCG-AgNPs showed lower virus titers in comparison to treatment with EGCG alone. After EGCG-AgNPs treatment, mucosal tissues showed a significant infiltration in dendritic cells and monocytes in comparison to NaCl-treated group, followed by significantly better infiltration of CD8+ T cells, NK cells and increased expression of IFN-α, IFN-γ, CXCL9 and CXCL10. (4) Conclusions: Our findings show that EGCG-AgNPs can become an effective novel antiviral microbicide with adjuvant properties to be applied upon the mucosal tissues.

Anti-HSV Activity of Metallic Nanoparticles Functionalized with Sulfonates vs. Polyphenols.

Metallic nanoparticles exhibit broad-spectrum activity against bacteria, fungi, and viruses. The antiviral activity of nanoparticles results from the multivalent interactions of nanoparticles with viral surface components, which result from the nanometer size of the material and the presence of functional compounds adsorbed on the nanomaterial surface. A critical step in the virus infection process is docking and entry of the virus into the host cell. This stage of the infection can be influenced by functional nanomaterials that exhibit high affinity to the virus surface and hence can disrupt the infection process. The affinity of the virus to the nanomaterial surface can be tuned by the specific surface functionalization of the nanomaterial. The main purpose of this work was to determine the influence of the ligand type present on nanomaterial on the antiviral properties against herpes simplex virus type 1 and 2. We investigated the metallic nanoparticles (gold and silver) with different sizes (5 nm and 30 nm), coated either with polyphenol (tannic acid) or sulfonates (ligands with terminated sulfonate groups). We found that the antiviral activity of nano-conjugates depends significantly on the ligand type present on the nanoparticle surface.

Functionalized Noble Metal Nanoparticles for the Treatment of Herpesvirus Infection.

Neuroinfections caused by herpesviruses, mainly by HHV-1, represent a significant problem for modern medicine due to the small number of therapeutic substances available in the pharmaceutical sector. Furthermore, HHV-1 infection has been linked to neurodegenerative processes such as Alzheimer's disease, which justifies the search for new effective therapies. The development of nanotechnology opens up new possibilities for the treatment of neuroinflammation. Gold and silver nanoparticles are gaining popularity, and the number of clinical trials involving metallic nanoparticles is constantly increasing. This paper reviews the research on gold and silver nanoparticles and their potential use in the treatment of herpesvirus neuroinfection.

Equid Alphaherpesvirus 1 (EHV-1) Influences Morphology and Function of Neuronal Mitochondria In Vitro.

Mitochondria are key cellular organelles responsible for many essential functions, including ATP production, ion homeostasis and apoptosis induction. Recent studies indicate their significant role during viral infection. In the present study, we examined the effects of equine herpesvirus type 1 (EHV-1) infection on the morphology and mitochondrial function in primary murine neurons in vitro. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). The organization of the mitochondrial network during EHV-1 infection was assessed by immunofluorescence. To access mitochondrial function, we analyzed reactive oxygen species (ROS) production, mitophagy, mitochondrial inner-membrane potential, mitochondrial mass, and mitochondrial genes' expression. Changes in mitochondria morphology during infection suggested importance of their perinuclear localization for EHV-1 replication. Despite these changes, mitochondrial functions were preserved. For all tested EHV-1 strains, the similarities in the increased fold expression were detected only for COX18, Sod2, and Tspo. For non-neuropathogenic strains (Jan-E and Rac-H), we detected mainly changes in the expression of genes related to mitochondrial morphology and transport. The results indicate that mitochondria play an important role during EHV-1 replication in cultured neurons and undergo specific morphological and functional modifications.

Role of Microglia in Herpesvirus-Related Neuroinflammation and Neurodegeneration.

Neuroinflammation is defined as an inflammatory state within the central nervous system (CNS). Microglia conprise the resident tissue macrophages of the neuronal tissue. Upon viral infection of the CNS, microglia become activated and start to produce inflammatory mediators important for clearance of the virus, but an excessive neuroinflammation can harm nearby neuronal cells. Herpesviruses express several molecular mechanisms, which can modulate apoptosis of infected neurons, astrocytes and microglia but also divert immune response initiated by the infected cells. In this review we also describe the link between virus-related neuroinflammation, and development of neurodegenerative diseases.

Equid Alphaherpesvirus 1 Modulates Actin Cytoskeleton and Inhibits Migration of Glioblastoma Multiforme Cell Line A172.

Equid alphaherpesvirus 1 (EHV-1) causes respiratory diseases, abortion, and neurological disorders in horses. Recently, the oncolytic potential of this virus and its possible use in anticancer therapy has been reported, but its influence on cytoskeleton was not evaluated yet. In the following study, we have examined disruptions in actin cytoskeleton of glioblastoma multiforme in vitro model-A172 cell line, caused by EHV-1 infection. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). Immunofluorescent labelling, confocal microscopy, real-time cell growth analysis and OrisTM cell migration assay revealed disturbed migration of A172 cells infected with the EHV-1, probably due to rearrangement of actin cytoskeleton and the absence of cell projections. All tested strains caused disruption of the actin network and general depolymerization of microfilaments. The qPCR results confirmed the effective replication of EHV-1. Thus, we have demonstrated, for the first time, that EHV-1 infection leads to inhibition of proliferation and migration in A172 cells, which might be promising for new immunotherapy treatment.

Lactoferrin-Functionalized Noble Metal Nanoparticles as New Antivirals for HSV-2 Infection.

(1) Background: Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In this work, we tested if silver and gold nanoparticles modified with lactoferrin (LF-Ag/AuNPs) can become novel microbicides with additional adjuvant properties to treat genital herpes infection. (2) Methods: The antiviral and cytotoxic activities of LF-Ag/AuNPs were tested in human skin HaCaT and vaginal VK-2-E6/E7 keratinocytes. Viral titers and immune responses after treatment with LF-Ag/AuNPs were tested in murine vaginal HSV-2 infection. (3) Results: LF-Ag/AuNPs inhibited attachment and entry of HSV-2 in human keratinocytes much better than lactoferrin. Furthermore, pretreatment with LF-AgNPs led to protection from infection. Infected mice treated intravaginally with LF-Ag/AuNPs showed lower virus titers in the vaginal tissues and spinal cords in comparison to treatment with lactoferrin. Following treatment, vaginal tissues showed a significant increase in CD8+/granzyme B + T cells, NK cells and dendritic cells in comparison to NaCl-treated group. LF-Ag/AuNPs-treated animals also showed significantly better expression of IFN-γ, CXCL9, CXCL10, and IL-1ß in the vaginal tissues. (4) Conclusions: Our findings show that LF-Ag/AuNPs could become effective novel antiviral microbicides with immune-stimulant properties to be applied upon the mucosal tissues.

Human herpesvirus type 2 infection of primary murine astrocytes causes disruption of the mitochondrial network and remodeling of the actin cytoskeleton: an in vitro morphological study.

Herpesviruses are capable of infecting not only neurons, where they establish latent infection, but also astrocytes. Since astrocytes are important for the functioning of the central nervous system (CNS), their infection may lead to serious neurological disorders. Thus, in the present study we investigated the ability of human herpesvirus type 2 (HHV-2) to infect primary murine astrocytes in vitro and the effect of infection on their mitochondrial network and actin cytoskeleton. In immunofluorescence assays, antibodies against HHV-2 antigens and glial fibrillary acidic protein (GFAP) were used to confirm that the infected cells are indeed astrocytes. Real-time PCR analysis showed a high level of HHV-2 replication in astrocytes, particularly at 168 h postinfection, confirming that a productive infection had occurred. Analysis of mitochondrial morphology showed that, starting from the first stage of infection, HHV-2 caused fragmentation of the mitochondrial network and formation of punctate and tubular structures that colocalized with virus particles. Furthermore, during the late stages of infection, the infection affected the actin cytoskeleton and induced formation of actin-based cellular projections, which were probably associated with enhanced intracellular spread of the virus. These results suggest that the observed changes in the mitochondrial network and actin cytoskeleton in productively infected astrocytes are required for effective replication and viral spread in a primary culture of astrocytes. Moreover, we speculate that, in response to injury such as HHV-2 infection, murine astrocytes cultured in vitro undergo transformation, defined in vivo as reactive astrocytosis.

Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes.

TLR3 provides immediate type I IFN response following entry of stimulatory PAMPs into the CNS, as it is in HSV infection. The receptor plays a vital role in astrocytes, contributing to rapid infection sensing and suppression of viral replication, precluding the spread of virus beyond neurons. The route of TLR3 mobilization culminating in the receptor activation remains unexplained. In this research, we investigated the involvement of various types of endosomes in the regulation of the TLR3 mobility in C8-D1A murine astrocyte cell line. TLR3 was transported rapidly to early EEA1-positive endosomes as well as LAMP1-lysosomes following stimulation with the poly(I:C). Later, TLR3 largely associated with late Rab7-positive endosomes. Twenty-four hours after stimulation, TLR3 co-localized with LAMP1 abundantly in lysosomes of astrocytes. TLR3 interacted with poly(I:C) intracellularly from 1 min to 8 h following cell stimulation. We detected TLR3 on the surface of astrocytes indicating constitutive expression, which increased after poly(I:C) stimulation. Our findings contribute to the understanding of cellular modulation of TLR3 trafficking. Detailed analysis of the TLR3 transportation pathway is an important component in disclosing the fate of the receptor in HSV-infected CNS and may help in the search for rationale therapeutics to control the replication of neuropathic viruses.

Disturbances of mitochondrial dynamics in cultured neurons infected with human herpesvirus type 1 and type 2.

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 ß-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.

Human herpesvirus type 1 and type 2 disrupt mitochondrial dynamics in human keratinocytes.

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.