Infected HeLa Cells Research Articles

Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

How Trypanosoma cruzi Feasts upon Its Mammalian Host

Trypanosoma cruzi has a complex relationship with its mammalian host in which parasite and host metabolic networks are intertwined. A genome-wide functional screen of T. cruzi infection in HeLa cells (Caradonna et al., 2013) divulges host metabolic functions and signaling pathways that impact intracellular parasite replication and reveals potential targets for therapeutic exploitation.

Rasgrp2 regulates the permissiveness of NIH3T3 cells to a herpes simplex virus 1 mutant with inactivated ICP34.5 gene

We have previously reported that mtHSV, a herpes simplex virus 1 (HSV-1) mutant with an inactivated gene for β-galactosidase, can efficiently lyse tumor but not normal cells. However, the mechanism of this selective oncolytic activity is so far unclear. In this study, using the phage display screening we identified the cellular protein binding to HSV-1 mutant (mtHSV) as (Ras guanyl releasing protein 2) Rasgrp2 which regulates the Ras signaling pathway. Rasgrp2 was found to bind directly to purified mtHSV as well as to mtHSV present within infected HeLa cells where it aggregated on the cell membrane. NIH3T3 cells were found nonpermissive to mtHSV but they became permissive following transformation with the Rasgrp2 gene. This effect was linked to the activation of the Ras-PKR signaling pathway. These observations indicate a key role of Rasgrp2 in the mtHSV infection of NIH3T3 cells and are important for the potential use of mtHSV in cancer therapy.

Cis-2-dodecenoic acid signal modulates virulence of Pseudomonas aeruginosa through interference with quorum sensing systems and T3SS

BackgroundCis-2-dodecenoic acid (BDSF) is well known for its important functions in intraspecies signaling in Burkholderia cenocepacia. Previous work has also established an important role of BDSF in interspecies and inter-kingdom communications. It was identified that BDSF modulates virulence of Pseudomonas aeruginosa. However, how BDSF interferes with virulence of P. aeruginosa is still not clear.ResultsWe report here that BDSF mediates the cross-talk between B. cenocepacia and P. aeruginosa through interference with quorum sensing (QS) systems and type III secretion system (T3SS) of P. aeruginosa. Bioassay results revealed that exogenous addition of BDSF not only reduced the transcriptional expression of the regulator encoding gene of QS systems, i.e., lasR, pqsR, and rhlR, but also simultaneously decreased the production of QS signals including 3-oxo-C12-HSL, Pseudomonas quinolone signal (PQS) and C4-HSL, consequently resulting in the down-regulation of biofilm formation and virulence factor production of P. aeruginosa. Furthermore, BDSF and some of its derivatives are also capable of inhibiting T3SS of P. aeruginosa at a micromolar level. Treatment with BDSF obviously reduced the virulence of P. aeruginosa in both HeLa cell and zebrafish infection models.ConclusionsThese results depict that BDSF modulates virulence of P. aeruginosa through interference with QS systems and T3SS.

Cowpox virus but not Vaccinia virus induces secretion of CXCL1, IL-8 and IL-6 and chemotaxis of monocytes in vitro

Orthopoxviruses are large DNA viruses which can cause disease in numerous host species. Today, after eradication of Variola virus and the end of vaccination against smallpox, zoonotic Orthopoxvirus infections are emerging as potential threat to human health. The most common causes of zoonotic Orthopoxvirus infections are Cowpox virus in Europe, Monkeypox virus in Africa and Vaccinia virus in South America. Although all three viruses are genetically and antigenically closely related, the human diseases caused by each virus differ considerably. This observation may reflect different capabilities of these viruses to modulate the hosts’ immune response. Therefore, we aimed at characterizing the specific cytokine response induced by Orthopoxvirus infection in vitro. We analysed the gene expression of nine human pro-inflammatory cytokines and chemokines in response to infection of HeLa cells and could identify an upregulation of cytokine gene expression following Cowpox virus and Monkeypox virus infection but not following Vaccinia virus infection. This was verified by a strong induction of especially IL-6, IL-8 and CXCL1 secretion into the cell culture supernatant following Cowpox virus infection. We could further show that supernatants derived from Cowpox virus-infected cells exhibit an increased chemotactic activity towards monocytic and macrophage-like cells. On the one hand, increased cytokine secretion by Cowpox virus-infected cells and subsequent monocyte/macrophage recruitment may contribute to host defence and facilitate clearance of the infection. On the other hand, given the assumed important role of circulating macrophages in viral spread, this may also point towards a mechanism facilitating delivery of the virus to further tissues in vivo.

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Aim There are many factors that are thought to contribute to T-cell immunodominance hierarchies including timing and levels of antigen presentation to T-cells. One of the most potent tools for studying antigen presentation for a specific determinant are monoclonal antibodies that are specific for not only MHC but a specific ligand, similar to a T cell receptor. In this study we utilize these antibodies to study levels and timing of antigen presentation of immunodominant (SVG9), sub-dominant (SLF9), and super sub-dominant (YTM9) T-cell epitopes during West Nile virus (WNV) infection. Methods High avidity antibodies specific for peptide/HLA-A∗02:01 complexes, SVG9, SLF9, and YTM9 were generated by mouse immunization with peptide/HLA complexes. Specific antibody producing hybridomas were expanded and the antibodies were purified from the cell supernatant. The purified antibodies were then used to stain WNV infected HeLa cells transfected with HLA-A∗02:01. Relative number of complexes was measured using flow cytometry. TAP inhibition studies were performed using TAP inhibitor protein ICP47. Results In this study, we demonstrate that viral peptide ligands are differentially expressed by A∗02:01 on the surface of a cell infected with WNV. Specifically, the super sub-dominant ligand (YTM9) was presented earlier and at 20 times the levels of the immunodominant (SVG9) and subdominant (SLF9) ligands. Transfection of ICP47 in infected cells show that all three epitopes are TAP dependent. Conclusions Using these highly specific reagents we show that there is differential ligand presentation within the restrictive allele A∗02:01. High levels of peptide HLA complexes on the surface of an infected do not correspond with immunodominance hierarchies as the super subdominant epitope YTM9 was presented at higher levels than the most immune dominant epitope SVG9. We then show that this differential presentation was not due the TAP dependence of the ligands.

NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

BackgroundRecently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV), the matrix (M) protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV.FindingIn order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10% at 48 h post-infection). In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells.ConclusionAlthough the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s) upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.

P141 Viral genome sequencing and small RNA detection by next generation sequencing

Introduction Next generation sequencing technologies are increasingly used in many domains. Their application to virology opens the door to various investigations such as virus sequencing and genotyping; analysis at quasispecies level; identification of causative agents of undiagnosed diseases; and finally study of host responses induced by viral infections. In this study, we used influenza- and rhinoviruses as models to validate some of these applications. Methods First we used influenza A virus infected cells to compare complete genome coverage after purification and ultra-deep sequencing of small RNAs (20–30 nt) or total fragmented RNAs (150–250 nt). Second, we sequenced small RNA population in rhinovirus (HRB-B14 and HRV-A39) infected HeLa cells and analyzed human micro RNA (miRNA) changes and the distribution of sequence reads on the viral genome. Results As expected the number of viral reads and genome coverage were much higher by sequencing total RNA, despite a lower ratio of viral versus host sequences. In contrast, small RNA purification from rhinovirus infected cells allowed us to point out to miRNA expression changes upon viral infection as well as over-expressed small viral RNAs (svRNAs). Conclusion svRNAs match the rhinovirus 5′UTR cloverleaf region, an important structural element implicated in the regulation of replication. The generation and the functional role of these svRNAs is currently under investigation.

Facteurs pronostiques de la cétoacidose en réanimation des urgences

Poliovirus obtained from mixedly infected HeLa cells (types 1 and 2) contains particles that are neutralizable by both type 1 and type 2 specific antisera. This alteration in phenotype is not transmitted to progeny virus and is believed to be due to phenotypic mixing. The progeny from most mixedly infected cells shows a high degree of phenotypic mixing (frequently 100%), and in some such cells there was no tangible evidence of the presence of one of the infecting genotypes. Mass yields from mixed infections produced the greatest proportion of phenotypic mixing in the first newly maturing virus.When HeLa cells are mixedly infected the amount of doubly neutralizable virus produced, the number of mixedly infected cells, and the number of cells producing phenotypically mixed virus all exceed theoretical expectations based on virus input by a factor of 2 to 14. Two factors are discussed which may contribute to this excess: one is the possible presence of HeLa cells which adsorb virus poorly or not at all and the second is a possible activating effect of plaque-forming on non-plaque-forming virus.

Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.

Host HDL biogenesis machinery is recruited to the inclusion ofChlamydia trachomatis-infected cells and regulates chlamydial growth

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that is the most common cause of sexually transmitted bacterial infections and is the etiological agent of trachoma, the leading cause of preventable blindness. The organism infects epithelial cells of the genital tract and eyelid resulting in a damaging inflammatory response. Chlamydia trachomatis grows within a vacuole termed the inclusion, and its growth depends on numerous host factors, including lipids. Although a variety of mechanisms are involved in the acquisition of host cell cholesterol and glycosphingolipids by C. trachomatis, none of the previously documented pathways for lipid acquisition are absolutely required for growth. Here we demonstrate that multiple components of the host high-density lipoprotein (HDL) biogenesis machinery including the lipid effluxers, ABCA1 and CLA 1, and their extracellular lipid acceptor, apoA-1, are recruited to the inclusion of C. trachomatis-infected cells. Furthermore, the apoA-1 that accumulates within the inclusion colocalizes with pools of phosphatidylcholine. Knockdown of ABCA1, which mediates the cellular efflux of cholesterol and phospholipids to initiate the formation of HDL in the serum, prevents the growth of C. trachomatis in infected HeLa cells. In addition, drugs that inhibit the lipid transport activities of ABCA1 and CLA 1 also inhibit the recruitment of phospholipids to the inclusion and prevent chlamydial growth.These results strongly suggest that C. trachomatis co-opts the host cell lipid transport system involved in the formation of HDL to acquire lipids, such as phosphatidylcholine, that are necessary for growth.

Orthobunyavirus Entry into Neurons and Other Mammalian Cells Occurs via Clathrin-Mediated Endocytosis and Requires Trafficking into Early Endosomes

La Crosse virus (LACV) is a leading cause of pediatric encephalitis and aseptic meningitis in the midwestern and southern United States, where it is considered an emerging human pathogen. No specific therapies or vaccines are available for LACV or any other orthobunyaviruses. Inhibition of LACV entry into cells is a potential target for therapeutic intervention, but this approach is limited by our current knowledge of the entry process. Here, we determined that clathrin-mediated endocytosis is the primary mechanism of orthobunyavirus entry and identified key cellular factors in this process. First, we demonstrated that LACV colocalized with clathrin shortly after infection in HeLa cells; we then confirmed the functional requirement of dynamin- and clathrin-mediated endocytosis for orthobunyavirus entry using several independent assays and, importantly, extended these findings to primary neuronal cultures. We also determined that macropinocytosis and caveolar endocytosis, both established routes of virus entry, are not critical for cellular entry of LACV. Moreover, we demonstrated that LACV infection is dependent on Rab5, which plays an important regulatory role in early endosomes, but not on Rab7, which is associated with late endosomes. These findings provide the first description of bunyavirus entry into cells of the central nervous system, where infection can cause severe neurological disease, and will aid in the design and development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system.

Coxsackie virus entry and spread in HeLa cells is aided by microvesicle release (170.22)

Abstract Microvesicles(MVs) released from plasma membrane expressing surface phosphatidylserine and ranging from 0.2-≤1 μm in diameter are reported to carry various membrane proteins, lipids and cytoplasmic components characteristic of the parental cell. Coxsackie virus B (CVB), a member of the enterovirus family is the main cause of meningitis and encephalitis in infants which may result in neurodevelopmental defects. Calpains are calcium-dependant cysteine proteases that degrade cytoplasmic and cytoskeletal proteins. They regulate a variety of actin-dependant cellular processes such as microvesiculation. CVB1 requires calpain activation for both entry and virus replication. Here, we show that knocking down calpain, using approaches such as small interfering RNA (siRNA), culminates in reduction of MV release, which then abrogates CVB1 entry and spread in HeLa cells. The calpain inhibitor calpeptin also caused similar reduction in CVB1 entry and spread to healthy target cells. Together, our findings provide evidence that CVB1 infected HeLa cells enhance MV production, and these MVs aid the spread of infection. Furthermore, inhibition of MV release using siRNA results in inhibition of CVB1 entry and spread.

Engineered phage-based therapeutic materials inhibit Chlamydia trachomatis intracellular infection

Developing materials that are effective against sexually transmitted pathogens such as Chlamydia trachomatis (Ct) and HIV-1 is challenging both in terms of material selection and improving bio-membrane and cellular permeability at desired mucosal sites. Here, we engineered the prokaryotic bacterial virus (M13 phage) carrying two functional peptides, integrin binding peptide (RGD) and a segment of the polymorphic membrane protein D (PmpD) from Ct, as a phage-based material that can ameliorate Ct infection. Ct is a globally prevalent human pathogen for which there are no effective vaccines or microbicides. We show that engineered phage stably express both RGD motifs and Ct peptides and traffic intracellularly and into the lumen of the inclusion in which the organism resides within the host cell. Engineered phage were able to significantly reduce Ct infection in both HeLa and primary endocervical cells compared with Ct infection alone. Polyclonal antibodies raised against PmpD and co-incubated with constructs prior to infection did not alter the course of infection, indicating that PmpD is responsible for the observed decrease in Ct infection. Our results suggest that phage-based design approaches to vector delivery that overcome mucosal cellular barriers may be effective in preventing Ct and other sexually transmitted pathogens.

Identification of genes contributing to the intracellular replication of Brucella abortus within HeLa and RAW 264.7 cells

Brucella abortus, the causative agent of brucellosis, can survive and replicate within host cells. Understanding bacterial virulence factors and bacteria–host cell interactions is critical for controlling brucellosis, yet very little is known about the virulence strategies and signaling pathways activated in phagocytes during infection to ensure their growth and survival. B. abortus was mutagenized by mini-Tn5Km2 transposon mutagenesis to identify virulence genes related to the internalization and intracellular replication of the bacteria. Of the total 2300 mutants used to infect HeLa cells, 23 mutants defective for intercellular growth and the mutated genes were identified. Sequence analysis of DNA flanking the transposon showed various insertion sites in bacterial genes that might be associated with virulence, including genes associated with lipoproteins, amino acid metabolism, translation, transcription, carbohydrate transport, coenzyme transport, inorganic ion transport, energy metabolism, membrane transport, and cell wall/membrane biogenesis. Moreover, mutants were classified into class I, class II and class III as higher, similar, and lower internalization, respectively, into HeLa cells. Furthermore, defective mutants for intracellular growth in HeLa cells were found to be defective in RAW 264.7 cells. Taken together, we suggest that the identified virulence associated genes might contribute to the intracellular growth and survival of B. abortus in phagocytes.

Vaccinia Mature Virus Fusion Regulator A26 Protein Binds to A16 and G9 Proteins of the Viral Entry Fusion Complex and Dissociates from Mature Virions at Low pH

Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.

Study on Survival of <I>Chlamydia trachomatis</I> in the Presence of Antichlamydial Drugs

Problem statement: Recurrent genital Chlamydia trachomatis infections due to treatment failures may results in complex sequelae leading to reproductive complexity and morbidity. It can be resulted by the heterotypic resistance with decreased drug susceptibility characteristic of the isolate. Studies are needed to understand the treatment failures and resistance characteristic of C. trachomatis. Hence, in vitro study was conducted on C. trachomatis isolate in the presence of antichlamydial drugs. Approach: Our aim was to study ygeD gene in C. trachomatis clinical isolate having decreased drug susceptibility profile and to analyze HeLa cells phenotypically upon infection in presence of antichlamydial drugs. Sequencing was done to check any mutational change (s) in ygeD gene of C. trachomatis isolate (CT-244), mRNA expression was analyzed in presence of antichlamydial drugs by Real Time RT-PCR. Transduction study was carried out in infected HeLa cells to detect changes at cellular level in presence of antichlamydial drugs by transducing with GFP/RFP-tagged proteins and analyzed by FACS. Results: A point mutation was detected in ygeD gene of C. trachomatis isolate. Further, mRNA expression level of ygeD gene was observed to be increased at 8 hpi in presence of doxycycline while in presence of azithromycin it was increased at 24 hpi. GFP-tagged plasma membrane protein expression in infected HeLa cells found to be reduced as compare to the uninfected cells. Upon infection, the RFP-tagged actin protein expression was up-regulated in comparison to the uninfected HeLa cells. No difference in expression of plasma membrane and actin protein was observed in susceptible serovar D and CT-244 isolate. Conclusion: The present study suggest that C. trachomatis isolate with decreased drug susceptibility profile may have an active efflux strategy for its survival in the presence of antichlamydial drugs and it may not affect its host cell plasma membrane or actin organization for its survival in order to resist the antichlamydial drugs.

Role of the Myristoylation Site in Expressing Exogenous Functional Proteins in Coxsackieviral Vector

We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.

Anti-Rhinovirus-specific Activity of the Alpha-sympathomimetic Oxymetazoline

Oxymetazoline (CAS 2315-02-8, OMZ, Nasivin) known as the active ingredient in nose drops and sprays demonstrates excellent efficacy in the treatment of rhinitis symptoms that are mainly caused by Rhinovirus infections. To elucidate possible modes of action, the antiviral activity of OMZ was studied in vitro on human pathogenic viruses. No in vitro effects were detected against enveloped RNA viruses, Parainfluenza Virus and Respiratory Syncytial Virus and against Adenovirus, a non-enveloped DNA-virus. In contrast, OMZ showed a specific inhibition of Human Rhinovirus (HRV). Analysis of production of HRV-14 and HRV-39 after treatment of infected HeLa cells using plaque-reduction assay and virus titration showed a strong dose-dependent antiviral activity of OMZ. Additional data demonstrated that OMZ did also directly affect HRV-14 infectivity in a dose-dependent manner. Analysis of a cell-protective effect of OMZ showed that pre-treatment of HeLa cells decreased virus adsorption as well as virus replication. Furthermore, OMZ induced a down-regulation of ICAM-1 expression on Tumor Necrosis Factor-alpha (TNF-alpha)-stimulated HeLa cells and human umbilical vein endothelial cells. Taken together, these results show that OMZ besides its vasoconstrictive action also possesses potent antiviral and anti-inflammatory activities. Therefore, OMZ does not only reduce rhinitis symptoms but additionally offers a causal therapeutic approach.

Sustained Cytoplasmic Delivery and Anti-viral Effect of PLGA Nanoparticles Carrying a Nucleic Acid-Hydrolyzing Monoclonal Antibody

Cytoplasmic delivery of a monoclonal antibody (mAb) with nucleic acid-hydrolyzing activity (3D8 scFv) using poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) was investigated for persistent anti-viral effect. 3D8 scFv-loaded PLGA (3D8-PLGA) NPs were prepared via a double emulsion method that was previously optimized. Flow cytometry and confocal microscopy was carried out to confirm the cellular uptake and cytoplasmic localization. immunochemical and fluorescence resonance energy transfer (FRET) assays tested the cytoplasmic release and hydrolyzing effect of 3D8 scFv, respectively. Anti-viral activity test was performed using MTT assay with vesicular stomatitis virus (VSV)-infected HeLa cells. 3D8-PLGA NPs were much more effectively taken into cells in dose- and time-dependent manner and localized in the cytosolic region, compared to free 3D8 scFv. 3D8 scFv was released and hydrolyzed RNAs in the cytoplasm, exhibiting the maxima at a period of time (12-24h). Anti-viral activity test revealed that 3D8-PLGA NP has dose- and time-dependent anti-viral effect and the maximum effect at the dose of 2mg/ml and the incubation of 3days. Cytoplasmic delivery of 3D8 scFv via PLGA NPs could enhance the viability of infected cells in sustained manner due to preserved activity, much improved cellular uptake and sustained release.