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.

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.

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.

SDAV, the Rat Coronavirus-How Much Do We Know about It in the Light of Potential Zoonoses.

Sialodacryoadenitis virus (SDAV) is known to be an etiological agent, causing infections in laboratory rats. Until now, its role has only been considered in studies on respiratory and salivary gland infections. The scant literature data, consisting mainly of papers from the last century, do not sufficiently address the topic of SDAV infections. The ongoing pandemic has demonstrated, once again, the role of the Coronaviridae family as extremely dangerous etiological agents of human zoonoses. The ability of coronaviruses to cross the species barrier and change to hosts commonly found in close proximity to humans highlights the need to characterize SDAV infections. The main host of the infection is the rat, as mentioned above. Rats inhabit large urban agglomerations, carrying a vast epidemic threat. Of the 2277 existing rodent species, 217 are reservoirs for 66 zoonotic diseases caused by viruses, bacteria, fungi, and protozoa. This review provides insight into the current state of knowledge of SDAV characteristics and its likely zoonotic potential.

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.

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.

Mechanisms of endocytosis utilized by viruses during infection.

Viruses, despite being relatively simple in structure and composition, have evolved a broad spectrum of mechanisms to exploit the host cell. To initiate effective infection, viruses or viral genomes have to enter cells. Recently studies have shown that apart from the direct fusion at the plasma membrane, endocytosis is more often the preferred means of entry into the host cell. Endocytosis is a complex phenomenon, that includes multiple pathways of membrane trafficking, such as clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis and phagocytosis. Endosomes offer a convenient and often rapid transit system across the plasma membrane and cytoplasm via the cellular microtubular network. They also provide protection to the virus from detection by the host's innate immune defences. What is important, viruses are able to utilize not just one, but multiple uptake routes. Identification of these processes and factors will not only allow a better insight into pathogenic mechanism, but may identify novel targets for future therapeutic development. This review provides insight on recent developments in the rapidly evolving field of viral entry.