Enterocin DD14 can inhibit the infection of eukaryotic cells with enveloped viruses.

Bacteriocins are ribosomally synthesized bacterial peptides endowed with antibacterial, antiprotozoal, anticancer and antiviral activities. In the present study, we evaluated the antiviral activities of two bacteriocins, enterocin DD14 (EntDD14) and lacticaseicin 30, against herpes simplex virus type 1 (HSV-1), human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero, Huh7 and Vero E6 cells, respectively. In addition, the interactions of these bacteriocins with the envelope glycoprotein D of HSV-1 and the receptor binding domains of HCoV-229E and SARS-CoV-2 have been computationally evaluated using protein-protein docking and molecular dynamics simulations. HSV-1 replication in Vero cells was inhibited by EntDD14 and, to a lesser extent, by lacticaseicin 30 added to cells after virus inoculation. EntDD14 and lacticaseicin 30 had no apparent antiviral activity against HCoV-229E; however, EntDD14 was able to inhibit SARS-CoV-2 in Vero E6 cells. Further studies are needed to elucidate the antiviral mechanism of these bacteriocins.

Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIß.

Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect was related to a cellular phosphatidylinositol 4-kinase type IIIß (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevented infection efficiently in acute cell models, eradicated infection in a chronic cell model, and lowered virus amounts in pancreas and heart in an acute mouse model. Altogether, instead of acting through the RAF/MEK/ERK pathway, vemurafenib affects the cellular PI4KB and, hence, enterovirus replication, opening new possibilities to evaluate further the potential of vemurafenib as a repurposed drug in clinical care. IMPORTANCE Despite the prevalence and medical threat of enteroviruses, presently, there are no antivirals against them. Here, we show that vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIß (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses.

Coxsackievirus-B4 Infection of Human Primary Pancreatic Ductal Cell Cultures Results in Impairment of Differentiation into Insulin-Producing Cells.

Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.

Emergence of Fluoxetine-Resistant Variants during Treatment of Human Pancreatic Cell Cultures Persistently Infected with Coxsackievirus B4.

This study reports the antiviral activity of the drug fluoxetine against some enteroviruses (EV). We had previously established a model of persistent coxsackievirus B4 (CVB4) infection in pancreatic cell cultures and demonstrated that fluoxetine could clear the virus from these cultures. We further report the emergence of resistant variants during the treatment with fluoxetine in this model. Four independent persistent CVB4 infections in Panc-1 cells were treated with fluoxetine. The resistance to fluoxetine was investigated in an acute infection model. The 2C region, the putative target of fluoxetine antiviral activity, was sequenced. However, Fluoxetine treatment failed to clear CVB4 in two persistent infections. The resistance to fluoxetine was later confirmed in HEp-2 cells. The decrease in viral titer was significantly lower when cells were inoculated with the virus obtained from persistently infected cultures treated with fluoxetine than those from susceptible mock-treated cultures (0.6 log TCID50/mL versus 4.2 log TCID50/mL, p < 0.0001). Some previously described mutations and additional ones within the 2C protein were found in the fluoxetine-resistant isolates. The model of persistent infection is an interesting tool for assessing the emergence of variants resistant to anti-EV molecules. The resistance of EV strains to fluoxetine and its mechanisms require further investigation.

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins.

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.

Bifidobacteria-derived lipoproteins inhibit infection with coxsackievirus B4 in vitro.

The aim of the present study was to investigate the potential of bifidobacteria in protecting cells from coxsackievirus B4 (CV-B4) infection. Bifidobacterial screening identified two of five strains that protected human epithelial type 2 (HEp-2) cell viability when bifidobacteria were incubated with viral particles prior to inoculation. In contrast, no effect was shown by incubating HEp-2 cells with bifidobacteria prior to CV-B4 inoculation. Cell wall lipoprotein aggregates (LpAs) secreted by the selected strains were assayed for their antiviral activity. The two LpAs exhibited antiviral activity when they were incubated with viral particles prior to inoculation of HEp-2 cells. Recombinant LpA-derived protein exhibited identical antiviral activity. To identify the peptide sequences interacting with the virus particles, LpA proteins were aligned with the peptide sequences of the north canyon rim and puff footprint onto coxsackievirus and adenovirus receptor (CAR). The in silico molecular docking study using CV-B3 as template showed low-energy binding, indicating a stable system for the selected peptides and consequently a likely binding interaction with CV-B. Bifidobacterium longum and Bifidobacterium breve peptides homologous to the viral north rim footprint onto CAR sequence formed hydrogen bonds with several viral residues in the north rim of the canyon, which were already predicted as interacting with CAR. In conclusion, proteins from bifidobacterial LpAs can inhibit infection with CV-B4, likely through binding to the capsid amino acids that interact with CAR.

Exposure of Piglets to Enteroviruses Investigated by an Immunoassay Based on the EV-G1 VP4 Peptide.

OBJECTIVE: The aim of this study was to investigate the exposure of piglets to enteroviruses-G (EV-G) through the presence of antibodies in their serum. METHODS: Serum samples were obtained from the vena cava of 10 piglets at 9 weeks of age and again 39 days later (day 39). They were tested using an immunoassay based on the EV-G1 VP4 peptide, since VP4 is highly conserved among the four Enterovirus capsid proteins, and by using a seroneutralization assay. RESULTS: For each serum collected on day 39 the optical density was high compared to the value obtained in serum collected earlier (p = 0.002). However, the titers of anti-EV-G1 serum neutralizing activity were not different in paired samples (p > 0.999). The sequence alignment of the EV-G1 VP4 peptide, encompassing 50 amino acids, used in the immunoassay showed 88% homology with EV-G, suggesting that antibodies directed toward other EV-G than EV-G1 may be detected. CONCLUSION: An immunoassay based on EV-G1 VP4 can detect an increased level of EV-G antibodies in piglet serum samples. Further studies are needed to determine whether this immunoassay may be useful for diagnosis and/or epidemiological studies and to monitor EV-G infection in pigs to evaluate strategies aimed to prevent enterovirus infections.

Coxsackievirus B4 Can Infect Human Peripheral Blood-Derived Macrophages.

Beyond acute infections, group B coxsackieviruses (CVB) are also reported to play a role in the development of chronic diseases, like type 1 diabetes. The viral pathogenesis mainly relies on the interplay between the viruses and innate immune response in genetically-susceptible individuals. We investigated the interaction between CVB4 and macrophages considered as major players in immune response. Monocyte-derived macrophages (MDM) generated with either M-CSF or GM-CSF were inoculated with CVB4, and infection, inflammation, viral replication and persistence were assessed. M-CSF-induced MDM, but not GM-CSF-induced MDM, can be infected by CVB4. In addition, enhancing serum was not needed to infect MDM in contrast with parental monocytes. The expression of viral receptor (CAR) mRNA was similar in both M-CSF and GM-CSF MDM. CVB4 induced high levels of pro-inflammatory cytokines (IL-6 and TNFα) in both MDM populations. CVB4 effectively replicated and persisted in M-CSF MDM, but IFNα was produced in the early phase of infection only. Our results demonstrate that CVB4 can replicate and persist in MDM. Further investigations are required to determine whether the interaction between the virus and MDM plays a role in the pathogenesis of CVB-induced chronic diseases.

Antibodies to VP1 of swine pasivirus in humans without evidence of transmission from a pig source.

BACKGROUND: Swine pasivirus (SPaV1) is a recently described enteric virus close to human parechoviruses and highly prevalent in pigs. Antibodies to Escherichia coli-expressed VP1 of SpaV1 have been found in a majority of humans in China. OBJECTIVES: The objectives were to estimate the antibody prevalence in a European country, to test if exposure to the virus was linked to pig products and if this exposure was a risk factor for the development of diabetes type 1. STUDY DESIGN: An ELISA test was developed and used to screen 842 healthy subjects with known exposure to pig products, 39 patients with diabetes type 1 and 20 controls. RESULTS: We identified a high seroprevalence (15.6%) reacting to VP1 of SPaV1 among healthy human subjects. Analysis of risk factors argues against cross-species transmission from pigs as the source of infection. Data also indicate that the presence of SPaV1 VP1-binding antibodies is not associated with diabetes type 1 in humans. CONCLUSION: Our results suggest that the seroreactivity frequently found in humans against SpaV1 is due to cross-reactivity with related antigen, perhaps a picornavirus, and that SpaV1 is not a zoonotic virus.

New Insights into the Understanding of Hepatitis C Virus Entry and Cell-to-Cell Transmission by Using the Ionophore Monensin A.

UNLABELLED: In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed that monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Using the selective pressure of monensin, we selected a monensin-resistant virus which has evolved to use a new entry route that is partially pH and clathrin independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, the Y297H mutation in E1 and the I399T mutation in hypervariable region 1 (HVR1) of E2, which confer resistance to monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In summary, we identified a mutation in HVR1 that overcomes the vesicular pH dependence, modifies the biophysical properties of particles, and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation. IMPORTANCE: Although several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to hepatic cells are still unclear. In our study, we used monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission pathways are both pH-dependent processes. We generated monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of hypervariable region 1 (HVR1) of the E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH dependency of cell-free and cell-to-cell transmission.

Persistent infection of human pancreatic cells with Coxsackievirus B4 is cured by fluoxetine.

Group B Coxsackieviruses (CVB) are involved in various acute clinical features and they can play a role in the development of chronic diseases like type 1 diabetes. The persistence of CVB has been described in vitro and in vivo in various models. Fluoxetine was reported to inhibit the replication of CVB1-3, which prompted us to study the in vitro antiviral activity of fluoxetine against CVB4 in models of acute infection. In addition we took advantage of a chronically CVB4-infected Panc-1 cell line to evaluate the antiviral effect of fluoxetine in a model of persistent CVB4 infection. An inhibition of the CVB4 replication was obtained when fluoxetine was added at 5.48µM to Hep-2 cell cultures. No inhibitory effect was observed when CVB4 was mixed with fluoxetine for 2h and filtered to eliminate fluoxetine before inoculation to cells, or when cells were treated up to 96h and washed before viral inoculation. Fluoxetine (5.48µM) reduced viral replication by more than 50% in acutely infected Panc-1 cell cultures. A dramatic decrease of infectious particles levels in supernatants of Panc-1 cells chronically infected with CVB4 was obtained a few days after treatment with fluoxetine and no infectious viral particle was found as soon as day 21 of treatment, and intracellular enteroviral RNA was undetectable by RT-PCR after three weeks of treatment. These data display that fluoxetine can inhibit the replication of CVB4 and can cure Panc-1 cells chronically infected with CVB4.

Serum-dependent enhancement of coxsackievirus B4-induced production of IFNα, IL-6 and TNFα by peripheral blood mononuclear cells.

Only a few reports have been published on the interactions between Coxsackievirus B4 (CVB4) and human peripheral blood mononuclear cells (PBMC) but have not been extensively documented. Human serum containing non-neutralizing anti-CVB4 antibodies increased CVB4-induced synthesis of IFNα by PBMC. In this study, we determined if CVB4 and human serum have the ability to activate inflammatory cytokines in addition to IFNα in PBMC cultures. PBMC from healthy donors were inoculated with infectious, inactivated CVB4 or with CVB4 incubated with dilutions of human serum or polyvalent IgG with anti-CVB4 activity. Levels of IFNα, TNFα, IL-6, IL-12, IFNγ and IL-10 in the cell-free supernatants of PBMC cultures were measured using ELISA. Infection was assessed by real-time PCR. PBMC inoculated with CVB4 produced inflammatory cytokines but not IFNα. When CVB4 was incubated with serum or IgG, IFNα was detected in the culture supernatants, and high concentrations of TNFα and IL-6 were measured. The concentrations of TNFα and IL-6 were not reduced in cultures inoculated with inactivated CVB4, whereas the IgG-dependent enhancement of IFNα, IL-6 and TNFα production with inactivated virus was suppressed. The potentiation of IFNα production was associated with a high intracellular viral load. Infectious and non-infectious CVB4 can induce the production of inflammatory cytokines but not IFNα by PBMC. High levels of IFNα, in addition to TNFα and IL-6, in culture supernatants were obtained when infectious CVB4 was combined with immune serum or IgG, and they were associated with high amounts of intracellular viral RNA.

Synergistic effect between colistin and bacteriocins in controlling Gram-negative pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial cells.

Pathogens resistant to most conventional antibiotics are a harbinger of the need to discover novel antimicrobials and anti-infective agents and develop innovative strategies to combat them. The aim of this study was to assess the in vitro activity of colistin alone or in combination with two bacteriocins, nisin A and pediocin PA-1/AcH, against Salmonella choleraesuis ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Yersinia enterocolitica ATCC 9610, and Escherichia coli ATCC 35150 (O157:H7). The strain most sensitive to colistin was enterohemorrhagic E. coli O157:H7, which was inhibited at a concentration of about 0.12 µg/ml. When nisin A (1.70 µg/ml) or pediocin PA-1/AcH (1.56 µg/ml) was combined with colistin, the concentrations required to inhibit E. coli O157:H7 were 0.01 and 0.03 µg/ml, respectively. The in vitro antigenotoxic effect of colistin was determined by using the comet assay method to measure the level of DNA damage in freshly isolated human peripheral blood leukocytes (PBLs) incubated with colistin for 1 h at 37°C. Changes in the tail extents of PBLs of about 69.29 ± 0.08 µm were observed at a final colistin concentration of about 550 ng/ml. Besides the synergistic effect, the combination of colistin (1 mg/ml) and nisin (2 mg/ml) permitted us to re-evaluate the toxic effect of colistin on Vero (monkey kidney epithelial) cells.

Persistent infection of thymic epithelial cells with coxsackievirus B4 results in decreased expression of type 2 insulin-like growth factor.

It has been hypothesized that a disturbance of central self-tolerance to islet ß cells may play a role in the enteroviral pathogenesis of type 1 diabetes. Whether enteroviruses can induce an impaired expression of ß-cell self-antigens in thymic epithelial cells has been investigated in a murine thymic epithelial (MTE) cell line. This cell line was permissive to the diabetogenic group B4 coxsackievirus (CV-B4) strain CV-B4 E2 and spontaneously expressed type 2 insulin-like growth factor (Igf2), the dominant self-antigen of the insulin family. In this model, a persistent replication of CV-B4 E2 was obtained, as attested to by the prolonged detection of intracellular positive- and negative-strand viral RNA by reverse transcription-PCR (RT-PCR) and capsid protein VP1 by immunofluorescent staining and by the release of infectious particles in culture supernatants. The chronic stage of the infection was characterized by a low proportion of VP1-positive cells (1 to 2%), whereas many cells harbored enteroviral RNA, as displayed by RT-PCR without extraction applied directly to a few cells. Igf2 mRNA and IGF-2 protein were dramatically decreased in CV-B4 E2-infected MTE cell cultures compared with mock-infected cultures, whereas housekeeping and interleukin-6 (Il6) gene expression was maintained and Igf1 mRNA was decreased, but to a lower extent. Inoculation of CV-B3, CV-B4 JVB, or echovirus 1 resulted in a low level of IGF-2 in culture supernatants as well, whereas herpes simplex virus 1 stimulated the production of the protein. Thus, a persistent infection of a thymic epithelial cell line with enteroviruses like CV-B4 E2 can result in a disturbed production of IGF-2, a protein involved in central self-tolerance toward islet ß cells.

Persistent infection of a carcinoma thyroid cell line with coxsackievirus B.

BACKGROUND: Viral infections are described as environmental factors that are implicated in various thyroid diseases. The role of enteroviruses (EV) in the pathogenesis of thyroid diseases has been suspected. Recently, we found that EV RNA could be detected in postoperative thyroid specimens. We decided to investigate the infection of a human thyroid cell line with coxsackievirus B4 (CVB4). METHODS: The wild-type human thyroid carcinoma cells K1 were inoculated with CVB4E2 at 2.1 x 10(7) TCID50/mL. The titer of the virus progeny was determined every 3 days on HEp-2 cells. CVB replication at the molecular level was monitored by searching for intracellular viral genomes using reverse transcription (RT)-polymerase chain reaction (PCR). EV VP1 capsid protein was detected by indirect immunocytofluorescence staining. Cell viability was determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) absorbance, and the nuclear morphology was assessed by Hoechst Dye staining. RESULTS: Infectivity assays with CVB4E2 revealed an increase in viral titers. Virus production decreased thereafter, but was not stopped by serial subculture for 24 days after infection. Detection of intracellular positive and negative viral RNA strands by RT-PCR was positive between days 1 and 14 postinfection (p.i.), and by semi-nested RT-PCR up to 24 days. K1 cell cultures infected with CVB4 were stained positively for EV VP1: the number of VP1-positive cells decreased rapidly within 6 days and remained low up to the end of culture. Compared with mock-infected cultures, viability in CVB4-infected cultures was around 100% up to 24 days. Cells with strongly fluorescent nuclei and/or fragmented nuclei were observed. CONCLUSION: We demonstrate for the first time that CVB4 could replicate in thyroid cells and could persist, with predominance of viral negative RNA strands for up to 24 days p.i. without obvious cytopathic effect. Our results suggest that CVB4 may lead to thyroid cell apoptosis. Further studies are needed to determine whether CVB could play a role in thyroid pathologies.