Human Herpesvirus 8 and Host-Cell Interaction: Long-Lasting Physiological Modifications, Inflammation and Related Chronic Diseases.

Oncogenic and latent-persistent viruses belonging to both DNA and RNA groups are known to cause serious metabolism alterations. Among these, the Human Herpesvirus 8 (HHV8) infection induces stable modifications in biochemistry and cellular metabolism, which in turn affect its own pathological properties. HHV8 enhances the expression of insulin receptors, supports the accumulation of neutral lipids in cytoplasmic lipid droplets and induces alterations in both triglycerides and cholesterol metabolism in endothelial cells. In addition, HHV8 is also known to modify immune response and cytokine production with implications for cell oxidative status (i.e., reactive oxygen species activation). This review underlines the recent findings regarding the role of latent and persistent HHV8 viral infection in host physiology and pathogenesis.

Human Herpesvirus 8 infection may contribute to oxidative stress in diabetes type 2 patients.

OBJECTIVE: To investigate the link between Human Herpesvirus 8 (HHV8) infection and plasma oxidative stress in patients with diabetes mellitus type 2 (DM2). RESULTS: Blood samples collected from DM2 and control subjects were screened for the presence of antibodies against HHV8 and for biomarkers of oxidative stress. We determined the products of radical damage on the plasma lipid fraction, such as malondialdehyde (MDA), fatty acid hydroperoxides (HP) and 7-ketocholesterol (7-keto), the oxidation products of unsaturated fatty acids (UFA) and cholesterol, respectively. The level of plasma antioxidant α-tocopherol (α-toc) was also assessed. Relevant differences were observed in the redox status in DM2 and either HHV8-positive or -negative control subjects. The level of α-toc significantly decreased in both DM2 and HHV8-positive subjects. Levels of MDA, HP and 7-keto were much higher in HHV8-positive and DM2 subjects, indicating that plasma oxidative stress is a common feature in both DM2 and HHV8-infection. In addition, 7-keto was further increased in HHV8-positive DM2 patients. We hypothesized that the HHV8-infection may contribute to the production of ROS, and hence to the oxidative stress closely related to the pathogenesis and development of DM2.

Anti-Human Herpesvirus 8 antibodies affect both insulin and glucose uptake by virus-infected human endothelial cells.

INTRODUCTION: Human Herpesvirus 8 (HHV8) is known to be the cause of the malignant tumour named Kaposi's sarcoma. It is believed to induce an intense modification of cell metabolism in endothelial cells. In this work we analysed the role of anti-HHV8 antibodies in both the insulin and glucose uptake of HHV8-infected primary human endothelial cells (HUVEC). METHODOLOGY: Western blotting, immunofluorescence and radiolabelled glucose were employed to assess the pPI3K expression, insulin binding and glucose-uptake by HUVEC cells, respectively. RESULTS: We confirmed that HHV8-infection is able to enhance both insulin binding and glucose-uptake in HHV8-infected primary endothelial cells; in addition, we found that anti-HHV8 specific antibodies are able to further increase both insulin and glucose uptake during the late latent phase of HHV8-infection in vitro. CONCLUSIONS: These findings suggest that a specific immune response to HHV8-infection may cooperate in boosting the cell metabolism, further enhancing the already increased insulin binding and glucose-uptake in HHV8-infected cells, which is a peculiar property of several oncogenic viruses.

Phenylpropenoids from Bupleurum fruticosum as Anti-Human Rhinovirus Species A Selective Capsid Binders.

The dichloromethane extract of the leaves of Bupleurum fruticosum was found to inhibit the replication of human rhinovirus (HRV) serotypes 14 and 39. Bioassay-guided fractionation led to the isolation of seven phenylpropenol derivatives (3-9), two polyacetylenes (1 and 2), and one monoterpene (10). Compounds 1 and 10 were identified as previously undescribed secondary metabolites after extensive 1D and 2D NMR experiments as well as high-resolution mass spectrometry. Compounds 2, 4, and 5 showed a selective inhibition of viral replication against HRV39 serotype, with 2 and 4 being the most active, with EC50 values of 1.8 ± 0.02 and 2.4 ± 0.04 µM. Mechanism of action studies indicated that 4 behaves not only as a capsid binder, interfering with the early phases of virus replication, but also as a late-phase replication inhibitor. Docking experiments were performed to confirm the ability of the antiviral phenylpropenoids to selectively fit into the hydrophobic pocket of VP1-HRV39.

Antimicrobial sulfonamides clear latent Kaposi sarcoma herpesvirus infection and impair MDM2-p53 complex formation.

Kaposi sarcoma herpesvirus (KSHV), also known as human herpesvirus 8, is the causative agent of Kaposi sarcoma; this malignant angiosarcoma is usually treated with conventional antitumor agents that can control disease evolution, but do not clear the latent KSHV episome that binds to cellular DNA. Some commercial antibacterial sulfonamides were tested for the ability to suppress latent KSHV. Quantitative PCR (qPCR) and cytofluorometry assays were used for detecting both viral DNA and the latency factor LANA (latency-associated nuclear antigen) in BC3 cells, respectively. The capacity of sulfonamides to impair MDM2-p53 complex formation was detected by an enzyme-linked immunosorbent assay method. The analysis of variance was performed according to one-way analysis of variance with Fisher as a post hoc test. Here we show that sulfonamide antibiotics are able to suppress the KSHV latent state in permanently infected BC3 lymphoma cells and interfere with the formation of the MDM2-p53 complex that KSHV seemingly needs to support latency and to trigger tumor cell transformation. These findings detected a new molecular target for the activity of sulfonamides and offer a new potential perspective for treating KSHV-induced lymphoproliferative diseases.

High Prevalence of Human Herpesvirus 8 Infection in Diabetes Type 2 Patients and Detection of a New Virus Subtype.

The prevalence of Human Herpesvirus 8 (HHV8) DNA and antiviral antibodies in Diabetes type 2 (DM2) and control subjects was studied, in order to confirm a possible link between DM2 and HHV8 infection. The HHV8-DNA from diabetic patients was typed for detecting possible genomic differences with known HHV8 reference viruses.DM2 patients and healthy controls were examined for the presence of HHV8 DNA into the peripheral blood lymphocytes. Both anti-lytic and latent phase antibodies were detected in HHV8 positive and negative diabetic patients, as well in a number of controls. The HHV8 ORF K1 and ORF 26 genes from DM2 patients were typed and matched to reference strains.A significant prevalence of HHV8 DNA in DM2 subjects versus healthy controls was detected (about 58 % against 27 %). Anti-lytic phase, but not anti-latent phase antibodies, were significantly increased in DM2 patients versus controls. In addition, about 30 % of HHV8 strains isolated from DM2 lymphocytes showed consistent differences in the ORF 26 gene sequence, so that a new HHV8 subtype was proposed. These findings give additional support to the hypothesis that HHV8 could be considered an additional risk factor for DM2 onset.

Combination of grape extract-silver nanoparticles and liposomes: A totally green approach.

In the present work, silver nanoparticles were prepared using a totally green procedure combining silver nitrate and an extract of grape pomace as a green source. Additionally, nanoparticles were stabilized using phospholipid and water and/or a mixture of water and propylene glycol (PG). To the best of our knowledge, grape-silver nanoparticle stabilized liposomes or PG-liposomes were formulated, for the first time, combining the residual products of wine-made industry, silver nitrate and phospholipids, avoiding the addition of hazardous substances to human health and the environment, in an easy, scalable and reproducible method. The structure and morphology of grape-silver nanoparticle stabilized vesicles were evaluated by transmission electron microscopy (TEM), UV-vis spectroscopy and photon correlation spectroscopy. Samples were designed as possible carrier for skin protection because of their double function: the grape extract acts as antioxidant and the colloidal silver as antimicrobial agent, which might be helpful in eliminating dangerous free radicals and many pathogenic microorganisms. Obtained nanoparticles were small in size and their combination with phospholipids did not hamper the vesicle formation, which were multilamellar and sized ~100nm. TEM images shows a heterogeneous distribution of nanoparticles, which were located both in the intervesicular medium and in the vesicular structure. Further, grape-silver nanoparticles, when stabilized by liposomes, were able to inhibit the proliferation of both Staphylococcus aureus and Pseudomonas aeruginosa and provided a great protection of keratinocytes and fibroblasts against oxidative stress avoiding their damage and death.

The Role of Human Herpesvirus 8 in Diabetes Mellitus Type 2: State of the Art and a Medical Hypothesis.

Diabetes is a common chronic disease due to an altered glucose metabolism, caused by the quantitative and/or qualitative dysfunction of the insulin hormone. Two types of diabetes are recognized: juvenile diabetes, or type 1, which has an autoimmune origin, and adult diabetes, or type 2 (DMT2), which covers 90-95 % of all diabetic patients.The causes of DMT2 are not yet clear: heredity, life style, nutrition, and environment are considered the main risk factors. Several viral infections, namely cytomegalovirus, coxsackie and other enteroviruses, rubella and hepatitis C virus, have been claimed to be associated with some forms of diabetes. The direct role of viruses as a cause or as a risk of type 1 diabetes has been amply described in several recent reviews. Therefore, this review focuses attention on the role of a human herpes pathogenic virus in the onset of DMT2. By carrying out an analysis of recent literature, we describe the findings reported on an extremely deceitful virus, such as Human Herpes virus 8, and present a medical hypothesis on a possible relationship between this virus and DMT2.

In vitro characterisation of a pleconaril/pirodavir-like compound with potent activity against rhinoviruses.

BACKGROUND: Rhinovirus infections do not only cause common colds, but may also trigger severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Even though rhinoviruses have been the focus of extensive drug development efforts in the past, an anti-rhinoviral drug still has to make it to the market. In the past, the viral capsid protein VP1 has been shown to be an important target for the development of antiviral molecules. Furthermore, many different chemical scaffolds appear to possess the properties that are required to inhibit virus replication by this mechanism of action. I-6602, an analogue of the rhinovirus inhibitor pirodavir, was previously identified as a potent inhibitor of rhinovirus infection. Here, we describe the antiviral activity of its analogue ca603, a molecule with a modified linker structure, and corroborate its mechanism of action as a capsid binder. FINDINGS: The molecule ca603 shows antiviral activity against a panel of rhino-and enteroviruses. Cross-resistance is observed against viruses with mutations that render them resistant to the inhibitory effect of the capsid binder pleconaril and thermostability assays demonstrate that the compound binds and stabilizes the viral capsid. Binding of the molecule to the VP1 protein is corroborated by in silico modeling. CONCLUSIONS: It is confirmed that ca603 inhibits rhinovirus replication by interaction with the VP1 protein and, by this, allows to further expand the chemical diversity of capsid-binding molecules.

Exploration of the anti-enterovirus activity of a series of pleconaril/pirodavir-like compounds.

BACKGROUND: The Enterovirus genus of the Picornaviridae is represented by several viral pathogens that are associated with human disease, namely Poliovirus 1, Enterovirus 71 and Rhinoviruses. Enterovirus 71 has been associated with encephalitis, while Rhinoviruses are a major cause of asthma exacerbations and chronic obstructive pulmonary disease. Based on the structure of both pleconaril and pirodavir, we previously synthesized some original compounds as potential inhibitors of Rhinovirus replication. METHODS: These compounds were explored for in vitro antiviral potential on other human pathogenic Enteroviruses, namely Enterovirus 71 on rhabdo-myosarcoma cells, Coxsackievirus B3 on Vero cells, Poliovirus 1 and Echovirus 11 on BGM cells. RESULTS: Activity was confirmed for compound against Rhinovirus 14. Furthermore, few compounds showed a cell-protective effect on Enterovirus 71, presented a marked improvement as compared to the reference drug pleconaril for inhibitory activity on both Enterovirus 71 and Poliovirus 1. The most striking observation was the clear cell protective effect for the set of analogues in a virus-cell-based assay for Echovirus 11 with an effective concentration (EC50) as low as 0.3 µM (Selectivity index or SI = 483), and selectivity indexes greater than 857 (EC50 = 0.6 µM) and 1524 (EC50 = 0.33 µM). CONCLUSION: Some of the evaluated compounds showed potent and selective antiviral activity against several enterovirus species, such as Enterovirus 71 (EV-A), Echovirus 11 (EV-B), and Poliovirus 1 (EV-C). This could be used as a starting point for the development of other pleconaril/pirodavir-like enterovirus inhibitors with broad-spectrum activity and improved effects as compared to the reference drugs.

Nutritionally Variant Streptococci Interfere with Streptococcus mutans Adhesion Properties and Biofilm Formation.

The bacterial species Streptococcus mutans is known as the main cause of dental caries in humans. Therefore, much effort has focused on preventing oral colonization by this strain or clearing it from oral tissues. The oral cavity is colonized by several bacterial species that constitute the commensal oral flora, but none of these is able to interfere with the cariogenic properties of S. mutans. This paper describes the interfering ability of some nutritionally variant streptococcal strains (NVS) with S. mutans adhesion to glass surfaces and also to hydroxylapatite. In mixed cultures, NVS induce a complete inhibition of S. mutans microcolony formation on cover glass slides. NVS can also block the adherence of radiolabeled S. mutans to hydroxylapatite in the presence of both saliva and sucrose. The analysis of the action mechanism of NVS demonstrated that NVS are more hydrophobic than S. mutans and adhere tightly to hard surfaces. In addition, a cell-free culture filtrate of NVS was also able to interfere with S. mutans adhesion to hydroxylapatite. Since NVS are known to secrete some important bacteriolytic enzymes, we conclude that NVS can be a natural antagonist to the cariogenic properties of S. mutans.

Neutral lipid alterations in human herpesvirus 8-infected HUVEC cells and their possible involvement in neo-angiogenesis.

BACKGROUND: Human Herpesvirus 8 (HHV8), the causative agent of Kaposi's sarcoma, induces an intense modification of lipid metabolism and enhances the angiogenic process in endothelial cells. In the present study, neutral lipid (NL) metabolism and angiogenesis were investigated in HHV8-infected HUVEC cells. The viral replication phases were verified by rtPCR and also by K8.1 and LANA immunostaining. RESULTS: Lipid droplets (Nile Red) were higher in all phases and NL staining (LipidTOX) combined with viral-antigen detection (immunofluorescence) demonstrated a NL content increase in infected cells. In particular, triglyceride synthesis increases in the lytic phase, whereas cholesteryl ester synthesis rises in the latent one. Moreover, the inhibition of cholesterol esterification reduces neo-tubule formation mainly in latently infected cells. CONCLUSIONS: We suggest that a reprogramming of cholesteryl ester metabolism is involved in regulating neo-angiogenesis in HHV8-infected cells and plays a likely role in the high metastatic potential of derived-tumours.

Autophagy activation and antiviral activity by a licorice triterpene.

The triterpene glycyrrhizic acid (GRA), the main product from the Glycyrrhiza glabra medicinal plant, is known for its antiinflammatory and antimicrobial activity. In this work, GRA was studied for its ability to induce the autophagic process activator Beclin 1 in epithelial cells and to observe how this property could influence its antiviral activity. After 24 h of treatment, GRA induced a Beclin 1 production that was more than twofold higher than that produced by rapamycin, used as a reference compound. When the compounds were added to HeLa cells together with the viruses, GRA demonstrated a strong antiherpes simplex virus type 1 (HSV1) activity, whereas rapamycin had no activity. However, if the compounds were added to the cells 24 h before the viruses, GRA induced the production of an even higher amount of Beclin 1 and showed an improved antiviral effect; under these conditions, rapamycin was also able to exert a significant anti-HSV1 activity. In conclusion, GRA is a strong inducer of the autophagy activator Beclin 1, which establishes a resistance state to HSV1 replication.

Synthesis of new compounds with promising antiviral properties against group A and B Human Rhinoviruses.

The human common cold, which is a benign disease caused by the Rhinoviruses, generally receives palliative symptomatic treatments, since no specific therapy against any of these viruses currently exists. In this work, some original synthetic compounds were produced and tested, in order to find non-toxic substances with an improved protection index (PI) for infected cells, as compared to reference drugs such as Pirodavir. We designed a series of novel molecules with a double oxygen in the central hydrocarbon chain and some modifications of the lateral methylisoxazole and propoxybenzoate moieties of lead compound 6602 (ethyl 4-{3-[2-(3-methyl-1,2-isoxazol-5-yl)ethoxy]propoxy}benzoate). It was found that most of these substances were actually less toxic than Pirodavir; in addition, the new molecule indicated as 8c was more than 30 times less toxic than Pirodavir, about twice as active on the group A strain of Rhinovirus HRV14, and even four times more effective on the group B strain HRV39, as compared to Pirodavir's PI.

Ultra-low power laser stimulation impairs the adhesion of Staphylococcus aureus to primary human cells, and interferes with the expression of staphylococcal pathogenic factors.

Lasers are commonly used in several fields of medicine as a complementary therapy for internal medicine, surgery and also diagnostics. The efficacy of ultra-low level laser therapy (ULLLT) at power levels around 0.15 mW/cm(2) has been demonstrated both in in vitro experiments and in the clinical environment. This work used an ULLLT laser source to analyze its efficacy on Staphylococcus aureus adhesion to cells and on its ability to produce pathogenic factors. Laser stimulation succeeded in impairing the binding of S. aureus to primary human cells in culture and in inhibiting the expression of coagulase, one of the main staphylococcal pathogenic factors. The importance of the extracellular matrix (ECM) and the modification of the ECM redox potential in these activities were also evidenced.

High prevalence of HHV8 infection and specific killer cell immunoglobulin-like receptors allotypes in Sardinian patients with type 2 diabetes mellitus.

The development of type 2 diabetes is thought to involve both environmental, possibly infectious, and genetic factors. Recently, a high prevalence of human herpesvirus 8 (HHV8) infection was observed in type 2 diabetes patients, and specific killer cell immunoglobulin-like receptors (KIR) allotypes were associated to both increased susceptibility to herpesvirus infection and risk to develop diabetes. However, no clear gene-disease or virus-disease associations have been established. To investigate the possible interplay between HHV8 infection, KIR allotype and type 2 diabetes, virus prevalence and KIR genotype were analyzed by PCR in 168 patients affected by type 2 diabetes and 108 control individuals belonging to the Sardinian population. Results showed a significant increase of HHV8 prevalence in type 2 diabetes patients versus controls (57% vs. 17%, P < 0.001), and a significant increase of KIR2DL2/DS2 homozygosity in diabetes patients infected with HHV8 compared to uninfected ones (64% vs. 14%, P < 0.0001), resulting in a significant OR of 11.31. In addition, the analysis of the frequency of the KIR2DL2/DS2 receptor and its HLA-C1 ligand, accordingly to the status of HHV8 infection, showed a significant increased correlation between KIR2DL2/DS2, type 2 diabetes and HLA-C1C1 genotype in the type 2 diabetes patients infected with HHV8 compared to uninfected ones (62% vs. 15%, P < 0.0001, OR = 8.64). These findings provide preliminary evidence that HHV8 infection might be a cofactor for type 2 diabetes in a specific subset of genetically susceptible individuals, and suggest the possibility that such patients might have an impaired immune-mediated component contributing to the development of type 2 diabetes.

Latent Herpesvirus 8 infection improves both insulin and glucose uptake in primary endothelial cells.

Human Herpesvirus 8 (HHV8), the causative agent of Kaposi sarcoma, induces a profound modification of infected cell behaviour, with reprogramming of gene expression and changes in physiological properties, over-expression of the insulin receptor, increased resistance to stress conditions and prolonged cell survival in conditions of serum deprivation. This paper shows that HHV8 infection induces a strong enhancement of both insulin and glucose uptake in primary endothelial cells (HUVEC). The increase in insulin uptake is already evident in the lytic phase of the viral infectious cycle, and reaches a maximum of up to 71% during the latent phase, whilst glucose uptake is slightly depressed during the lytic viral infection, but significantly enhanced compared with the control during the latent phase of viral infection, with an average increase of about 37% 25 days after cell infection.

An internalin a probe-based genosensor for Listeria monocytogenes detection and differentiation.

Internalin A (InlA), a protein required for Listeria monocytogenes virulence, is encoded by the inlA gene, which is only found in pathogenic strains of this genus. One of the best ways to detect and confirm the pathogenicity of the strain is the detection of one of the virulence factors produced by the microorganism. This paper focuses on the design of an electrochemical genosensor used to detect the inlA gene in Listeria strains without labelling the target DNA. The electrochemical sensor was obtained by immobilising an inlA gene probe (single-stranded oligonucleotide) on the surfaces of screen-printed gold electrodes (Au-SPEs) by means of a mercaptan-activated self-assembled monolayer (SAM). The hybridisation reaction occurring on the electrode surface was electrochemically transduced by differential pulse voltammetry (DPV) using methylene blue (MB) as an indicator. The covalently immobilised single-stranded DNA was able to selectively hybridise to its complementary DNA sequences in solution to form double-stranded DNA on the gold surface. A significant decrease of the peak current of the voltammogram (DPV) upon hybridisation of immobilised ssDNA was recorded. Whole DNA samples of L. monocytogenes strains could be discriminated from other nonpathogenic Listeria species DNA with the inlA gene DNA probe genosensor.

Study of the biological activity of novel synthetic compounds with antiviral properties against human rhinoviruses.

Picornaviridae represent a very large family of small RNA viruses, some of which are the cause of important human and animal diseases. Since no specific therapy against any of these viruses currently exists, palliative symptomatic treatments are employed. The early steps of the picornavirus replicative cycle seem to be privileged targets for some antiviral compounds like disoxaril and pirodavir. Pirodavir's main weakness is its cytotoxicity on cell cultures at relatively low doses. In this work some original synthetic compounds were tested, in order to find less toxic compounds with an improved protection index (PI) on infected cells. Using an amino group to substitute the oxygen atom in the central chain, such as that in the control molecule pirodavir, resulted in decreased activity against Rhinoviruses and Polioviruses. The presence of an -ethoxy-propoxy- group in the central chain (as in compound I-6602) resulted in decreased cell toxicity and in improved anti-Rhinovirus activity. This compound actually showed a PI >700 on HRV14, while pirodavir had a PI of 250. These results demonstrate that modification of pirodavir's central hydrocarbon chain can lead to the production of novel derivatives with low cytotoxicity and improved PI against some strains of Rhinoviruses.