Cytopathic Cell Research Articles

Surface air gas discharge plasma: An ecofriendly virus inactivation approach to enhance CPRRs mediated antiviral genes expression against airborne bio-contaminant (human Coronavirus-229E)

Emerging bio-contaminants (airborne viruses) exploits and manipulate host (human) metabolism to produce new viral particles, evading the host's immune defences and leading to infections. Non-thermal plasma, operating at atmospheric pressure and ambient temperature, is explored for virus inactivation, generating RONS that interact and denatures viral proteins. However, various factors affecting virus survival influence the efficacy of non-thermal plasma. Glucose analogue 2-DG, a metabolic modifier used in this study, disrupts the glycolysis pathway viruses rely on, creating an unfavourable environment for replication. Here, airborne HCoV-229E bio-contaminant was treated with plasma for inactivation, and the presence of RONS was analysed. Metabolically altered lung cells were subsequently exposed to the treated airborne viruses. Cytopathic effect, spike protein, and cell death were evaluated via flow cytometry and confocal microscopy, and CPRRs mediated antiviral gene expression was evaluated using PCR. Gas plasma-treated viruses led to reduced virus proliferation in unaltered lung cells, although few virus particles survived the exposure, as confirmed by biological assessment (cytopathic effects and live/dead staining). A combination approach of gas plasma-treated viruses and altered lung cells displayed drastic virus reduction compared to the control group, established through confocal microscopy and flow cytometry. Furthermore, altered lung cell enhances gene transcription responsible for innate immunity when exposed to the gas plasma-treated virus, thereby impeding airborne virus propagation. This study demonstrates the significance of a surface air gas plasma and metabolic alteration approach in enhancing genes targeted towards antiviral innate immunity and tackling outbreaks of emerging bio-contaminants of concerns (airborne viruses).

CRISPR activation as a platform to identify interferon stimulated genes with anti-viral function.

Interferon Stimulated Gene (ISG) expression plays a key role in the control of viral replication and development of a robust adaptive response. Understanding this dynamic relationship between the pathogen and host is critical to our understanding of viral life-cycles and development of potential novel anti-viral strategies. Traditionally, plasmid based exogenous prompter driven expression of ISGs has been used to investigate anti-viral ISG function, however there are deficiencies in this approach. To overcome this, we investigated the utility of CRISPR activation (CRISPRa), which allows for targeted transcriptional activation of a gene from its endogenous promoter. Using the CRISPRa-SAM system to induce targeted expression of a panel of anti-viral ISGs we showed robust induction of mRNA and protein expression. We then employed our CRISPRa-SAM ISG panel in several antiviral screen formats to test for the ability of ISGs to prevent viral induced cytopathic cell death (CPE) and replication of Dengue Virus (DENV), Zika Virus (ZIKV), West Nile Virus Kunjin (WNVKUN), Hepatitis A Virus (HAV) and Human Coronavirus 229E (HCoV-229E). Our CRISPRa approach confirmed the anti-viral activity of ISGs like IFI6, IFNβ and IFNλ2 that prevented viral induced CPE, which was supported by high-content immunofluorescence imaging analysis. This work highlights CRISPRa as a rapid, agile, and powerful methodology to identify and characterise ISGs and viral restriction factors.

Synthesis and evaluation of alkoxy-substituted enamides against influenza A virus in vitro and in vivo

Alkoxy-substituted enamides are often used as synthetic intermediates due to their special reactivity. To the best our knowledge, the biological activity of alkoxy-substituted amines has never been reported so far. We have synthesized a series of alkoxy-substituted enamides to study their anti-influenza A virus activity in vitro and in vivo. Among these compounds, compound E-2o had the best antiviral activity (EC50 = 2.76 ± 0.67 μM) and low cytotoxicity (CC50 = 662.87 ± 24.85 μM). The mechanism of action of this compound was preliminarily explored by us. It alleviated the cytopathic effects and cell death caused by different subtypes of influenza A virus. Different drug delivery methods and timed dosing experiments had shown that E-2o had the best therapeutic effect and mainly played a role in the early stages of virus replication. The expansion of influenza viruses in cells was inhibited by reducing ROS accumulation, cell apoptosis, and autophagy. Alkoxy-substituted enamide E-2o reduced the production of interferon and other pro-inflammatory factors in the RIG-Ⅰ pathway and its downstream NF-κB was induced by influenza A virus in vitro and in vivo. It avoided damage in the mice which was caused by excessive inflammatory factors. In addition, the weight loss and lung lesion damage in mice caused by influenza virus were improved by compound E-2o. Therefore, Alkoxy-substituted enamide E-2o could inhibit the replication of influenza viruses in vivo and in vitro, and has the potential to be developed into a drug for treating influenza.

Effective inhibition of HCoV-OC43 and SARS-CoV-2 by phytochemicals in vitro and in vivo

Several coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus OC43 (HCoV-OC43), can cause respiratory infections in humans. To address the need for reliable anti-coronavirus therapeutics, we screened 16 active phytochemicals selected from medicinal plants used in traditional applications for respiratory-related illnesses. An initial screen was completed using HCoV-OC43 to identify compounds that inhibit virus-induced cytopathic effect (CPE) and cell death inhibition. Then the top hits were validated in vitro against both HCoV-OC43 and SARS-CoV-2 by determining virus titer in cell supernatant and virus-induced cell death. Finally, the most active phytochemical was validated in vivo in the SARS-CoV-2-infected B6.Cg-Tg(K18-ACE2)2Prlmn/J mouse model. The phytochemicals lycorine (LYC), capsaicin, rottlerin (RTL), piperine and chebulinic acid (CHU) inhibited HCoV-OC43-induced cytopathic effect and reduced viral titres by up to 4 log. LYC, RTL and CHU also suppressed virus replication and cell death following SARS-CoV-2 infection. In vivo, RTL significantly reduced SARS-CoV-2-induced mortality by ∼40% in human angiotensin-converting enzyme 2 (ACE2)-expressing K18 mice. Collectively, these studies indicate that RTL and other phytochemicals have therapeutic potential to reduce SARS-CoV-2 and HCoV-OC43 infections.

Патологическая анатомия поражения сосудов легких при COVID-19

Pathological anatomy of pulmonary vascular lesions plays a key role in understanding the patho-genesis and morphogenesis of COVID-19. This review focuses on morphological features of pulmonary vascular injury in COVID-19. The virus is known to be capable of causing not only cytopathic cell damage of various organs and tissues (type II pneumocytes, cardiomyocytes, neurons, epithelial cells of the gas-trointestinal tract) but also endotheliotropic damage that aggravates the development and course of acute alveolar damage to the lungs. We studied the literature on microscopic changes in the vessel walls in lung tissue for 2020–2022. This review discusses the issues of local or systemic vascular lesions, morphologi-cal changes in the vessels over time, the likelihood of the development of microangiopathy, vasculitis, and endotheliitis in COVID-19 patients. Particular attention is paid to possible mechanisms of endotheliopathy in COVID-19 patients and its role in the microthrombi genesis. We speculate that in the future, postmor-tem lung examination in COVID-19 patients using histological, histochemical, and immunohistochemical methods will clarify the features of alterative and inflammatory changes and reparative processes in the blood vessel walls, as well as neoangiogenesis at different stages of the disease. Keywords: pathological anatomy, COVID-19, lungs, endotheliopathy, endotheliitis, microthrombus formation

Human Kidney Spheroids and Monolayers Provide Insights into SARS-CoV-2 Renal Interactions.

Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death

BackgroundA key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6–8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection.MethodsTo examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo.ResultsBCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma.ConclusionsThese findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

Features of SARS-COV-2 infection and directions of drug and vaccine creation

Features of SARS-COV-2 infection and directions of drug and vaccine creation

Real-Time Visualization and Quantification of Oncolytic M1 Virus In Vitro and In Vivo

Alphavirus M1 is a promising oncolytic virus for cancer therapy. Here, we constructed a fluorescent reporter virus for real-time visualization and quantification of M1 virus both in vitro and in vivo. The reporter-encoding M1 virus maintained the characteristics of parental virus in the aspects of structure, replication capacity, the feature to induce cytopathic cell death, and the property of tumor targeting. The fluorescence is positively correlated with virus replication both in vitro and in vivo. More importantly, the reporter can be stably expressed for at least 10 generations in a serial passage assay. In summary, we successfully constructed stable and authentic reporter viruses for studying M1 virus and provided a feasible technical route for gene modification of oncolytic virus M1.

Kidney Involvement in COVID-19: Need for Better Definitions

Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus outbreak in China at the end of 2019, increasing interest emerged regarding renal involvement during coronavirus disease 2019 (COVID-19). AKI with or without proteinuria is described in a variable percentage of patients with COVID-19, and several reports outlined an increased mortality risk in those with AKI. The mechanisms of renal injury during COVID-19 are difficult to study due to the interference of several coexisting factors, such as polypharmacy, hypoxia, and cytokine storm. Just like severe acute respiratory syndrome coronavirus, SARS-CoV-2 virus entry into target cells is facilitated by the presence of angiotensin-converting enzyme 2 (ACE2) expressed in respiratory cells as well as renal tubular cells and podocytes, making the hypothesis of a direct renal infection particularly intriguing. Farkash et al.1 observed renal tubular vacuolization and virus-like inclusions in tubular cells by electron microscopy (EM) in the autopsy of a patient with COVID-19. Similar EM findings together with suggestive immunohistochemical stain for SARS-CoV-2 proteins in tubules were shown in a few patients from two independent Chinese COVID-19 autopsy cohorts (Diao B, Wang C, Wang R, Feng Z, Tan Y, Wang H, et al. [2020] Human kidney is a target for novel severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infection. 10.1101/2020.03.04.20031120).2 However, the same immunohistochemical was not considered specific when used by others.3 Of note, when specific and highly sensitive molecular methods for tissue viral identification were used (i.e., RNAscope3 or real-time PCR,4 the presence of SARS-CoV-2 RNA could not be demonstrated within renal tissue, and none of the aforementioned autopsy studies included viral RNA isolation in the kidney. Interestingly, viral nephropathies with direct tubular infection and viral replication within epithelial cells (i.e., BK virus nephropathy) are usually associated with substantial viruria due to the direct release of viral particles in the tubular lumen following cytopathic cell injury. So far, there is no solid evidence of a correlation between urinary levels of SARS-CoV-2 viral copies and the degree of kidney injury in patients with COVID-19.5 Moreover, the presence of viral particles by pure morphologic evaluation (EM) does not demonstrate by itself a direct cytopathic effect nor a replicative potential because it only supports viral entry. More specific morphologic correlates of viral cytopathy (nuclear atypia/dysmorphology and syncytia) have not been shown yet. Three cases of collapsing glomerulopathy during SARS-CoV-2 infection have been reported,6 presenting with severe kidney injury and variable proteinuria: in genetically predisposed subjects, increased levels of cytokines (i.e., IFNs) in patients with COVID-19 are thought to drive podocyte injury, resulting in collapsing glomerulopathy as can be observed in other viral-associated nephropathies. Although in one case, the presence of virus-like particles in podocytes was observed by EM, the hypothesized mechanism of injury does not require direct kidney infection. Taken together, these observations suggest that caution should be used when addressing the etiology of kidney dysfunction in patients with COVID-19 because unequivocal evidence of direct viral infection of the kidney is still lacking in these severe and acutely ill patients. Disclosures All authors have nothing to disclose. Funding None.

Anti-viral activity of Zingiber officinale (Ginger) ingredients against the Chikungunya virus

Chikungunya is one of the highly infectious viral disease without vaccine and anti-viral. Aim of present study is to check the anti-chikungunya activities of Zingiber officinale (Ginger) in the animal cell culture model. The medicinal plant extract was prepared from Z. officinale rhizome. Median tissue culture infective dose (TCID50) of Chikungunya virus (CHIKV) and Maximum non-toxic dose (MNTD) of Z. officinale extract was determined in Vero cell-line on the basis of cell viability followed by MTT assay. In vitro anti-chikungunya activity was performed in Vero cell-line with MNTD and half of MNTD of Z. officinale medicinal plant extract. The anti-viral effect of Z. officinale was studied by observing the cytopathic effects and cell viability measured by MTT assay. Maximum non-toxic dose of Z. officinale plant extract was found 62.5 μg/ml. During anti-chikungunya experimentation, cell viability increased to 51.05% and 35.10%, when Vero cells were pre-treated with MNTD and half of MNTD of Z. officinale extract respectively. Similarly, in co-treatment, when MNTD, half of MNTD of Z. officinale and Median tissue culture infective dose CHIKV were inoculated simultaneously, then the viability of Vero cell-line was increases by 52.90% and 49.02% respectively. The rhizome extracts of Z. officinale have high potential to treat CHIKV. Medicinal plants and their metabolites are most important sources of antimicrobial and can be utilized for the development of new drugs. In view of the rapid expansion of CHIKV at the global level, there is an urgent need to develop newer anti-chikungunya drugs.

Oncolytic activity of HF10 in head and neck squamous cell carcinomas

Recent developments in therapeutic strategies have improved the prognosis of head and neck squamous cell carcinoma (HNSCC). Nevertheless, 5-year survival rate remains only 40%, necessitating new therapeutic agents. Oncolytic virotherapy entails use of replication-competent viruses to selectively kill cancer cells. We aimed to explore the potential of HF10 as an oncolytic virus against human or mouse HNSCC cell lines, and primary-cultured HNSCC cells. HF10 replicated well in all the HNSCC cells, in which it induced cytopathic effects and cell killing. Next, we investigated the oncolytic effects of HF10 in ear tumor models with human or mouse tumor cells. We detected HF10-infected cells within the ear tumors based on their expression of green fluorescent protein. HF10 injection suppressed ear tumor growth and prolonged overall survival. In the syngeneic model, HF10 infection induced tumor necrosis with infiltration of CD8-positive cells. Moreover, the splenocytes of HF10-treated mice released antitumor cytokines, IL-2, IL-12, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha, after stimulation with tumor cells in vitro. The HF10-treated mice that survived their original tumor burdens rejected tumor cells upon re-challenge. These results suggested that HF10 killed HNSCC cells and induced antitumoral immunity, thereby establishing it as a promising agent for the treatment of HNSCC patients.

Measuring the Infectious Titer of Recombinant Adenovirus Using Tissue Culture Infection Dose 50% (TCID50) End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR).

Traditionally, adenovirus and recombinant adenovirus infectious titers have been measured by plaque assay, in which the cells are infected with serially diluted adenovirus stock and then overlaid with agar; a plaque will form as the result of a single infectious event. Although this method gives a quantitative readout (number of plaques corrected for the dilution), there can be issues with sensitivity and reproducibility, especially when adenovirus serotypes are used that infect standard cell lines with poor efficiency. An alternative approach is to plate serial dilutions of the cells growing in the wells of a 96-well tissue culture plate and determine the dilution at which 50% of the wells are infected. This ancient and reliable technique known as the "tissue culture infection dose 50%" (TCID50) end-point dilution method has been used for titering a number of viruses, especially those that do not readily form plaques. Usually, infected wells are determined by direct examination for cytopathic effect (CPE) or cell viability. However, by combining a 96-well TCID50 format and the power of quantitative polymerase chain reaction (qPCR) for detection, a large increase in sensitivity-in our hands 10-fold, with a range of both transgenes and adenovirus serotypes-can be achieved. This protocol uses a 96-well TCID50 format, in conjunction with qPCR for sensitive and quantitative positive-well calling, to determine infectious titer of adenovirus vectors.

Antiviral potentials of Lactobacillus plantarum, Lactobacillus amylovorus, and Enterococcus hirae against selected Enterovirus.

Enteroviruses have been associated with a host of clinical presentations including acute flaccid paralysis (AFP). The site of primary replication for most enteroviruses is the gastrointestinal tract (GIT) and lactic acid bacteria (LAB) may confer protection in the GIT against them. This study therefore investigates the antiviral potential of some selected lactic acid bacteria against enterovirus isolates recovered from AFP cases. The antiviral activities of Lactobacillus plantarum, Lactobacillus amylovorus, and Enterococcus hirae in broth culture, their cell-free supernatant (CFS), and bacterial cell pellets were assayed against Echovirus 7 (E7), E13, and E19 in a pre- and post-treatment approach using cytopathic effect (CPE) and cell viability (MTT) assay. The tested Lactobacillus plantarum, Lactobacillus amylovorus, and Enterococcus hirae strains have good antiviral properties against E7 and E19 but not against E13. Lactobacillus amylovorus AA099 shows the highest activity against E19. The pre-treatment approach displays better antiviral activities compared to post-treatment approach. The LAB in broth suspension have better antiviral activities than their corresponding CFS and bacterial pellet. Lactic acid bacteria used in this study have the potential as antiviral agents.

Preclinical Testing of an Oncolytic Parvovirus in Ewing Sarcoma: Protoparvovirus H-1 Induces Apoptosis and Lytic Infection In Vitro but Fails to Improve Survival In Vivo

About 70% of all Ewing sarcoma (EWS) patients are diagnosed under the age of 20 years. Over the last decades little progress has been made towards finding effective treatment approaches for primarily metastasized or refractory Ewing sarcoma in young patients. Here, in the context of the search for novel therapeutic options, the potential of oncolytic protoparvovirus H-1 (H-1PV) to treat Ewing sarcoma was evaluated, its safety having been proven previously tested in adult cancer patients and its oncolytic efficacy demonstrated on osteosarcoma cell cultures. The effects of viral infection were tested in vitro on four human Ewing sarcoma cell lines. Notably evaluated were effects of the virus on the cell cycle and its replication efficiency. Within 24 h after infection, the synthesis of viral proteins was induced. Efficient H-1PV replication was confirmed in all four Ewing sarcoma cell lines. The cytotoxicity of the virus was determined on the basis of cytopathic effects, cell viability, and cell lysis. These in vitro experiments revealed efficient killing of Ewing sarcoma cells by H-1PV at a multiplicity of infection between 0.1 and 5 plaque forming units (PFU)/cell. In two of the four tested cell lines, significant induction of apoptosis by H-1PV was observed. H-1PV thus meets all the in vitro criteria for a virus to be oncolytic towards Ewing sarcoma. In the first xenograft experiments, however, although an antiproliferative effect of intratumoral H-1PV injection was observed, no significant improvement of animal survival was noted. Future projects aiming to validate parvovirotherapy for the treatment of pediatric Ewing sarcoma should focus on combinatorial treatments and will require the use of patient-derived xenografts and immunocompetent syngeneic animal models.

Anti-apoptotic Protein BIRC5 Maintains Survival of HIV-1-Infected CD4+ T Cells

HIV-1 infection of CD4+ Tcells leads to cytopathic effects and cell demise, which is counter to the observation that certain HIV-1-infected cells possess a remarkable long-term stability and can persist lifelong in infected individuals treated with suppressive antiretroviral therapy (ART). Using quantitative mass spectrometry-based proteomics, we showed that HIV-1 infection activated cellular survival programs that were governed by BIRC5, a molecular inhibitor of cell apoptosis that is frequently overexpressed in malignant cells. BIRC5 and its upstream regulator OX40 were upregulated in productively and latently infected CD4+ Tcells and were functionally involved in maintaining their viability. Moreover, OX40-expressing CD4+ Tcells from ART-treated patients were enriched for clonally expanded HIV-1sequences, and pharmacological inhibition of BIRC5 resulted in a selective decrease of HIV-1-infected cells invitro. Together, these findings suggest that BIRC5 supports long-term survival of HIV-1-infected cells and may lead to clinical strategies to reduce persisting viral reservoirs.

High oncogenic risk human papillomavirus and urinary bladder cancer

To determine the role of human papillomavirus (HPV) of high oncogenic risk in the development of urinary bladder cancer. 100 patients (72 men and 28 women) aged 38 to 90 years (mean age 65+/-10 years) diagnosed with bladder cancer were examined and underwent treatment. Clinical assessment was complemented by enzyme-linked immunosorbent assays for the presence of antiviral antibodies to herpes simplex virus (HSV) type 1 and type 2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), urethra scraping for detecting high oncogenic risk HPV. Tumor tissue was sampled for PCR virus detection. Semi-quantitative analysis was used to evaluate the components of lymphocyte-plasmocyte and leukocyte infiltrates and cytopathic changes in tumor tissue. There were positive correlations between cytopathic cell changes (koylocytosis and intranuclear inclusions, as manifestations of HPV) and the level of antiviral antibodies, the presence of viruses in the tumor, as well as with the components of the lymphoid-plasmocyte infiltrate. Negative correlations were found between the presence of papillomatosis and the above changes. Human papillomavirus is believed to be a trigger for the initiation of a tumor in young patients with a latent infection (CMV and EBV, HSV, HPV). Cytopathic changes (kylocytosis and intranuclear inclusions) were associated with the activity and morphological features of herpes-viral infections. Their degree varied depending on the stage of the process, but not on the anaplasia degree. Papillomatosis is associated with a more favorable course of the tumor process.

Idarubicin is a broad-spectrum enterovirus replication inhibitor that selectively targets the virus internal ribosomal entry site.

Enterovirus 71 (EV71) causes life-threatening diseases with neurological manifestations in young children. However, the treatment of EV71 infections remains an unmet medical need. Idarubicin (IDR) is an anthracycline compound that is used therapeutically for certain types of tumour. In this study, we identified IDR as an EV71 inhibitor, which displayed antiviral potency in the submicromolar range and substantially protected cells from the cytopathic effects and cell death caused by EV71 infections. The antiviral effects extended to several other enterovirus (EV) species, and these effects were independent of cytotoxicity or topoisomerase inhibition. Structure-activity relationship studies indicated the importance of the anthracycline scaffold for anti-EV potency. IDR effectively blocked the synthesis of viral protein and RNA, but not the viral proteolysis processes. Moreover, anthracyclines were demonstrated to suppress EV internal ribosomal entry site (IRES)-mediated translation; conversely, the cellular p53 IRES activity was not sensitive to IDR action. Inhibition of IRES-mediated translation by IDR correlated with the affinity of binding between IDR and the particular IRES. Moreover, IDR impaired binding between the EV71 IRES RNA and hnRNP A1, a known host IRES trans-acting factor. In sum, we have identified a USA FDA-approved anticancer drug with the new indication as a selective EV IRES binder and inhibitor. The finding may also provide leads for the development of novel antiviral therapies directed at the EV IRES RNA.

Human MicroRNA-602 Inhibits Hepatitis C Virus Genotype 1b Infection and Promotes Tumor Suppressor Gene Expression in a Hepatoma Cell Line

Objective: Hepatitis C virus (HCV) infects 200 million people worldwide, inducing cirrhosis and hepatocellular carcinoma. Liverspecific microRNA (miR) miR-122 facilitates HCV replication, and Locked Nucleic Acid (LNA)-anti-miR-122 was used successfully as a mono-therapy in HCV infected patients. Studies show that anti-miR-122 may negatively impact hepatocyte metabolism. Previously, we showed that human (hsa) miR-602 relative intracellular expression was up-regulated in microarray profiling when transfected with HCV genotype 1b, and hsa-miR-602 lowered HCV accumulation in infected Huh-7.5 cells. This study compares the effectiveness of hsa-miR-602 and LNA-anti-miR-122 on inhibiting HCV replication and regulating the expression of tumor suppressor genes p53, p73, and RASSF1A. Methods: Huh-7.5 cells were transfected with HCV genotype 1b and treated with hsa-miR-602, LNA-anti-miR-122 or both. Cytopathic effect (CPE) and cell aggregation were observed regularly after transfection. HCV load and localization in the un-transfected, transfected, and treated cells were detected by qRT-PCR and immunostaining, respectively. qRT-PCR detected the expression of p53, p73, and RASSF1A. The Cancer Cell Lines Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) were used to correlate gene copy number and expression for TP53, TP73 and RASSF1A in normal and hepatocellular carcinoma (HCC) patient tissues and HCC cell lines. The survival curve for patients with MIR- 602 expression was done using TCGA HCC clinical data sets. Results: HCV load decreased in all treated cells but was more pronounced with hsa-miR-602. HCV transfected Huh-7.5 cells treated with hsa-miR-602 showed no morphological changes whereas cells treated with LNA-anti-miR-122 or LNA-anti-miR-122 and hsa-miR-602 together showed morphological changes. p53, p73, RASSF1A were down-regulated in HCV transfected cells and all treatments restored expression to near normal levels. Similar down-regulated expression was seen in the HCC patient samples and cell lines. Conclusion: Hsa-miR-602 effectively reduces HCV load and restores p53, p73, and RASSF1A expression in HCV transfected cells.

EFFECT OF SELENIUM AND /OR VITAMIN E ON BOVINE HERPES TYPE 1 INFECTION IN VITRO CULTURED CELLS

Bovine herpes virus type 1 (BHV-1) still the causes of great economic lossesin the livestock industry and trade because there are no available drug that proved to be fully effective against it. In this study, the antioxidant of selenium and/ or the vitamin E were evaluated as a natural anti-viral drugs. The antiviral activities against Bovine Herpesvirus type 1 (BHV-1) were evaluated by the reduction of the viral cytopathic effect 1nMadin-Darby Bovine Kidney cell line (MDBK) with, GSH (glutathione reduced) and MAD (malondialdehyde) assays. Selenium in concentrations 0.01 and 0.1 µM and / or Vit. E in concentrations 50 and 100 µM with two concentrations were added at different stages of the viral infection (pre and post treatment). This study concluded that the higher concentration of Selenium and Vit. E (0.1 µM+100µM respectively) reduced the Cytopathic Effect (CPE) of BHV-1 1n 50% and increased GSH level as well as decreased MDA level, in pre infection treatment assay.