The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. 1st are subtle symptoms contagious with the HSV1(Oral) virus? The extract from neem bark (NBE) significantly blocked HSV-1 entry into cells at concentrations ranging from 50 to 100 μg/ml. RNA-binding protein G-rich sequence factor 1 (GRSF1) was identified as a new target of miR-101; GRSF1 binds to HSV-1 p40 mRNA and enhances its expression, facilitating viral proliferation. Having been washed with phosphate buffered saline, 50 plaque-forming units (PFU) of HSV-1 was added to each well. glabra. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step.
They appeared as wedges or angular segments of closed spherical capsids, the angle ranging from less than 30 degrees to greater than 270 degrees. * The most reliable ways to avoid transmission of sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV), are to abstain from sexual activity or to be in a long-term mutually monogamous relationship with an uninfected partner. Upon reactivation from latency, viruses are produced in neuronal cells and transported back to the epithelia to cause recurrent lesions. L’une des caractéristiques de l’infection virale herpès est la capacité du virus de persister à l’état latent dans les ganglions sensitifs après la primo-infection, avec des réactivations récurrentes provoquées par de nombreux stimuli, qui peuvent causer des lésions cliniquement apparentes. To date, none of the currently approved antivirals can eliminate an established latent infection. When genital herpes symptoms appear, it’s usually 2 to 14 days after a person is exposed to the virus. Screening all 117,649 combinations for the most desirable combination is an enormous task in terms of labor and time.
In the present work, we demonstrate that the hydrophobic polycation N,N-dodecyl, methyl-polyethylenimine (DMPEI) (previously determined to be maximally antimicrobial when used as a non-covalent coating (10,12,13,20)) can also decrease the infectivity of HSV-1 or -2. A number of platforms have been developed to amplify nucleic acids at a single temperature, thus alleviating the need for thermal cycling equipment –. All chemicals and reagents were from Sigma-Aldrich Chemical Co. It is a multi-step process that is initiated by specific interaction of viral envelope glycoproteins and host cell surface receptors (Spear, 1993; Spear et al., 2000). GRSF1 belongs to a family of RNA-binding proteins called the heterogeneous nuclear ribonucleoprotein F/H protein family (hnRNP F/H)16, which includes hnRNP F, hnRNP H, hnRNP H3, hnRNP H2 and GRSF117. Polyethylene sheets, from McMaster Carr (Atlanta, GA), were cut into 2.5×2.5-cm slides. Non-lubricated male latex condoms (Trojan®), distributed by Church & Dwight Co.
The human oral SCC cell line SAS was obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan). They were rinsed thoroughly in double-distilled (dd) H2O and cut into 3×4-cm rectangles for further use. The MTS assay kit (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay) was purchased from Promega (Madison, WI). Most of the membrane-bound isoforms can bind through their cytoplasmic tails to the PDZ domain in l-afadin. La détection d’anticorps IgG permet la détermination du statut immunologique du patient et fournit la preuve sérologique d’une exposition antérieure au virus herpès. The peptide composed entirely of d-amino acids (TAT-Cd) was equally as effective as the l-amino acid version, indicating that chirality is not critical (9). DMPEI was synthesized from linear 217-kDa polyethylenimine (PEI) as previously described (15).
Finally, we also included tumor necrosis factor (TNF)-α,22 a cellular protein that induces activation of nuclear factor kappa B (NF-κB) and cellular death pathways. Briefly, 1 g of 750-kDa branched PEI was dissolved in 15 mL of anhydrous methanol, and an excess (17.2 mL) of iodomethane was added to the solution at room temperature. We chose this assay because detection of HIV-1 proviral DNA is an established method for early infant diagnosis , and the HIV-1 DNA RPA assay used here has been well-characterized elsewhere . The solvent was then removed by rotary evaporation, and the resultant solid was dissolved in ddH2O and dialyzed against it four times, followed by lyophilization to obtain solid PMPEI. The bark of neem plant [Azardirachata indica Linn (Meliaceae)] has been widely used as a traditional medicine for many centuries in tropical countries. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) revealed that HSV-1 ICP4 induced hsa-mir-101-2 expression by directly binding to its promoter region. Coated condoms were prepared similarly, except that DMPEI was dissolved in butanol (23).
HSV-1 KOS strain and HSV-2 186/syn+-1 strain were originally obtained from Priscilla A. Cells were exposed to ultrasound in the presence or absence of microbubbles at room temperature. The viruses were diluted with an aqueous buffer (phosphate-buffered saline (PBS) supplemented with 1% BCS, 0.01% glucose). Vero cells were from the American Type Culture Collection (#CCL-81, Manassas, VA) and maintained in supplemented cell culture DMEM (described above) at 37°C in a 5% CO2 atmosphere. Ca2+ switch protocol.Cells were grown in DME containing 2 mM Ca2+ with 10% FCS (NC medium). To determine the antiviral effect of DMPEI against HSV-1 and HSV-2, a DMPEI-coated polyethylene slide was placed in a polystyrene Petri dish (6.0×1.5-cm), and a 10-μL droplet of either HSV-1 ((4.4±3.9)×105 plaque forming units (PFU)) or HSV-2 ((5.0±0.69)×104 PFU) was added to the middle of the coated region. In the present study, we report that three cationic β-peptides, peptides 1 to 3 (Fig.
After 15 min at room temperature, the slides were separated and washed thoroughly with 0.99 mL of the supplemented PBS buffer. Phosphate buffered saline (PBS) was from EMD (Rockland, MA). The infectious viral titer from the above experiments was compared to those of a control with two uncoated slides and a control with the virus never in contact with slides to determine the relative reduction in viral titer upon incubation with DMPEI-coated slides (9,12,15). To analyze the data for each body location, the average temperature over time was calculated for each volunteer. Specifically, cell growth medium was removed from confluent monolayers of Vero cells in a 6-well plate and replaced with 0.5 mL of diluted virus in each well. G. These data indicate that the upregulation of miR-101 by HSV-1 infection may contribute to the activation of hsa-mir-101-2 precursor expression.
Plaques were counted to determine the viral titer of the solutions. HSV-1 (10 μL containing (7.8±0.40)×107 PFU) was incubated with 0.99 mL of either branched 750-kDa PEI (2 mg/mL) or PMPEI (1 mg/mL) in the supplemented PBS buffer for 30 min on a rotating arm. Ten microliters of a 5 mg/mL MTT (Sigma, St. DMPEI was dissolved in dimethylsulfoxide at 10 mg/mL and added to 3 volumes of ddH2O with vigorous stirring. The resultant mixture was lyophilized and resuspended in ddH2O with vigorous stirring and sonication until the suspension was devoid of visible chunks. The stained cells were photographed with an Olympus microscope (model CK2), equipped with a 35-mm camera, controlled by the Olympus automatic exposure photographic system (PM-10AK3). A DMPEI suspension thickened with hydroxyethyl-cellulose (HEC) was prepared by making a 1.5% solution of HEC in 2.5 mL of PBS containing the desired concentration of DMPEI.
Viral titers were determined by plaque assay on Vero cells. A 3 mg/mL DMPEI suspension in PBS was 10-fold serially diluted, followed by incubation of 100 μL thereof for 1 h with confluent monolayers of Vero cells seeded in a 96-well plate. HSV-1 infection on an NIH 3T3 fibroblast cell line was used as an in vitro model system to search for new therapeutic drug combinations. The MTS assay was carried out according to the manufacturer’s instructions. Each 50 µL reaction contained 29.5 µL rehydration buffer, 2.1 µL biotin-labeled forward primer (5′-[biotin]-TGGCAGTATTCATTCACAATTTTAAAAGAAAAGG-3′), 2.1 µL reverse primer (5′-CCCGAAAATTTTGAATTTTTGTAATTTGTTTTTG-3′), 0.6 µL FAM-labeled nfo probe (5′-[FAM]-TGCTATTATGTCTACTATTCTTTCCCCTGC[dSpacer]CTGTACCCCCCAATCCCC[C3 Spacer]-3′), 3.2 µL water, one enzyme pellet, 2.5 µL magnesium acetate, and 10 µL of water or HIV-1 DNA template. Thereafter, the virus and suspension mixture was filtered with a 0.45-μm cut-off filter (Pall Life Sciences), and the filtrate was 10-fold serially diluted and used in an infectivity assay to determine the reduction in viral titer. Increasing concentration of (10–6400 μg/ml) of the NBE were added to monolayers of Vero cells in triplicate and the plates were incubated at 37°C in a 5% CO2.
After 12 h, ICP4 overexpression greatly enhanced luciferase activity compared to the control group (). Specifically, 0.5 mL of a DMPEI suspension (or the supplemented PBS buffer) and 0.5 mL containing 210±10 PFU of HSV-1 were mixed in a T25 flask and gently agitated for 1 h at 37°C. Then the virus and DMPEI suspension mixture was removed and replaced with 5 mL of DMEM containing 1% BCS and 0.16% human IgG. Cells were exposed to ultrasound and viability was evaluated using the MTT assay. For antiviral assays with various concentrations of DMPEI suspensions containing 1.5% HEC, 25 μL of either HSV-1 ((3.8±0.20)×105 PFU) or HSV-2 ((2.5±0.90)×104 PFU) was added to 2.5 mL of HEC-thickened DMPEI suspensions in 20-mL scintillation vials containing 1-cm long magnetic stir bars (to aid in mixing). Vials were placed on a rotating arm for 30 min, after which time 0.5 mL of the solution was carefully withdrawn by a pipette and diluted for the subsequent infectivity assay. 1H) as previously described (1).
To assess the antiviral activity of the coated latex condoms, the above-described procedure for polyethylene slides was followed, except that a piece of coated condom replaced the coated slide. Background activity was measured in mock-infected cultures and was subtracted from the data. After a 15-min incubation with HSV-1 ((8.8±0.80)×105 PFU) or HSV-2 ((4.8±0.1)× 104 PFU), the plain slide was separated from the condom piece, and both were submerged in a 50-mL falcon tube containing 10 mL of the supplemented PBS buffer. Second, if TNF-α had no antiviral effect or enhanced HSV-1 infection, we sought to determine whether it would be screened out of the possible drug combinations by the FSC technique. The same experiment was performed with a piece of uncoated latex condom and also without one as controls. Ten microliters of diluted product was then added to the sample pad of the lateral flow strip, and the strip was placed in a well of a 96-well plate containing an excess of running buffer. Exposure to a DMPEI-coated polyethylene slide reduced the viral titer to below the limit of detection of the plaque assay, thus yielding at least a 5-log reduction in viral titer for HSV-1 and at least a 4-log reduction for HSV-2 compared to minimal reduction for the uncoated polyethylene slide ().
After rinsing with PBS, 1.5 ml of 1.0 mg/ml X-gal in ferricyanide buffer was added to each well and the blue color developed in the cells was examined. The biotin probe was incubated with nuclear extracts from HeLa cells transfected with pICP4-FLAG, the constructs were separated by electrophoresis. Since HSVs are transmitted by direct contact with viral lesions, an antiviral formulation should ideally be available in a form that can intimately interact with infected tissues. Because DMPEI was deliberately designed as a non-leaching surface coating and it is insoluble in aqueous solution (10), we explored whether its water-soluble homolog, per-methylated PEI (PMPEI), or even an underivatized PEI were capable of inactivating HSV-1 (as a soluble form of the polymer could be easily administered to a lesion). To examine this possibility, SAS cells were firstly exposed to ultrasound with microbubbles for 10 s. Not only was the polycation PMPEI incapable of lowering the HSV-1 titer by more than a single log in aqueous solution, but also it was less potent than the unalkylated PEI (). We concluded, therefore, that the polycationic nature was not the sole, or even the main, determinant of anti-HSV activity and substantial hydrophobicity was required as well.
Mammalian nectin-1 is much more highly conserved in sequence than are nectin-2 (9, 23, 29) and HVEM (14, 22). The heights … The cultures were then incubated at 37°C in serum-supplemented DMEM in the presence and absence of various concentrations of β-peptides and assayed for β-d-galactosidase activity 6 h later. To test this hypothesis, we prepared an aqueous suspension of DMPEI and investigated whether it was capable of inactivating HSV-1 and HSV-2. The fourth module was the search algorithm, which provided the next set of stimulant dosages for directing the biocomplex system toward the desired phenotype (). Based on an MTS assay, we observed a 50% cell cytotoxicity (CC50) at a 120-μg/mL polycation concentration. Several methods were tested for securing tubes to the body to allow convenient incubation of RPA reactions in the axilla.
Since the DMPEI filtrate exhibited no visible toxicity (results not shown), we concluded that toxicity must result from particles larger than approximately 0.45-μm. The activation of the reporter luciferase gene as a measure of cell fusion was examined using reporter lysis assay (Promega) at 24 hr post mixing. To confirm whether miR-101 directly binds to the GRSF1 3′UTR and negatively regulates its gene expression, the GRSF1 3′UTR or a mutant 3′UTR () was cloned downstream of an EGFP gene in a pcDNA3 vector. Moreover, most of the observed antiviral activity was due to the DMPEI particles larger than 0.45 μm because incubation with DMPEI suspension filtrate did not have a marked anti-HSV effect (). Lastly, filtering itself did not contribute to a reduction of viral titer (second bar, ). Initially, glycoprotein gB and gC bind to heparan sulfate proteoglycans on the cell surface, attaching the virus to the host cell. Reduction of viral titer of HSV-1 incubated for 30 min at room temperature in a buffered aqueous solution (the left bar), in that subsequently filtered through a 0.45-μm filter (the middle left bar), in that containing 0.3 mg/mL of DMPEI suspension …
If used as a therapeutic, DMPEI would presumably be applied topically in the presence of both the virus and host cells. This localization and subsequent redistribution are indistinguishable from those observed for human nectin-1 (compare with Fig. During this experiment, we observed a dose-dependent response for the DMPEI suspension with regard to both inhibition of HSV-1 infection and toxicity. Control cells received medium only. As the concentration of the DMPEI in the suspension was increased to 0.1, 0.3, and 0.5 mg/mL, we observed a 85%, 95%, and 100% decrease in viral titer, respectively (). Treatment of HSV-1-infected cells for 16 hours with this drug combination resulted in less than 0.1% GFP-positive cells, suggesting that it completely blocked HSV-1 infection. From these data, the half-maximal inhibitory concentration (IC50) was calculated to be 24 μg/mL.
4C), and in the Houston summer sun (Fig. For a DMPEI suspension to be used as a topical therapeutic, it should be “pharmaceutically elegant”, i.e., in the form of a homogeneous cream or lotion. Recently we showed that RPE cells express gD receptor nectin-1 to allow HSV-1 entry (Tiwari et al., 2008). pGRSF1-FLAG, pshR-GRSF1 were transfected into cells and incubated for 24 h, followed by HSV-1 infection. Note that a control aqueous solution containing the same concentration of HEC alone had no appreciable influence on viral titer. Reduction of viral titers of HSV-1 (a) and HSV-2 (b) incubated for 30 min at room temperature in an aqueous PBS buffer (the left set of bars), in that thickened with 1.5% HEC (the 2nd set of bars), and in that thickened with 1.5% HEC in which various … However, the plaque number was increased three-fold by exposure to ultrasound in presence of microbubbles; therefore the increase was attributed to the virus entering cells through pores in the plasma membrane.
As seen in , more than a 3-log reduction in infectivity for both viruses compared to the uncoated latex condom was observed. Reduction of viral titers of HSV-1 (a) and HSV-2 (b) incubated for 15 min at room temperature in a buffered aqueous solution (the left set of bars), in that sandwiched between an uncoated polyethylene slide and an uncoated piece of latex condom (the middle … This antibody completely inhibited the enhanced binding of HSV-1 gD:Fc to MDCK-nectin-1 cells depleted for Ca2+ whereas the anti-nectin-2 antibody was without effect (Fig. More than a 3-log reduction was observed with a stretched DMPEI-coated latex condom for HSV-1 and more than a 2-log reduction for HSV-2 (). β-Peptide 2 can form a globally amphiphilic 14-helix, but this molecule, lacking ACHC residues, does not adopt the 14-helical conformation in aqueous solution, and peptide 2 does not enter HeLa cells efficiently (46). In addition, stretching may produce cracks in the DMPEI coating where HSVs can remain unmolested. However, side effects for high doses of ribavirin are a drawback of this drug.
We have demonstrated herein that the hydrophobic polycationic material DMPEI could be employed both in a prophylactic modality (as a coating on latex condoms) and in a therapeutic modality (as a suspension) to inactivate HSVs. This method may be modified for convenience, as reactions may be incubated at several body locations and secured using available materials that may be reused for many experiments. Although we previously observed no appreciable acute toxicity, either in vitro (23) or in vivo (21,26) for DMPEI coatings, a suspension presumably results in a more intimate contact between the cells and the hydrophobic polycation and thus greater toxicity. We next determined whether the inhibitory activity of NBE on HSV-1 entry was attributed to target cells or viral particles. GRSF1 protein binds to the 5′UTR of the influenza virus nucleocapsid gene transcript and promotes the translation of viral mRNAs to facilitate viral replication in infected cells14,30. As to a possible prophylactic use, both optimized noncovalent painting of, and covalent attachment of hydrophobic polycations to, latex (polyisoprene) condoms should be explored and the resultant coated condoms tested in terms of their long-term stability and safety. This research was supported by the U.S.
In the present study, we examined cells immediately after exposure to ultrasound and detected depressed spots on the cell surface, especially after exposure to ultrasound with microbubbles. AML is a recipient of a Martin Family graduate fellowship. 5. Expression of β-galactosidase signals the entry of virus into cells and expression of viral and reporter genes. Herpes simplex viruses. •, peptide 1; ○, peptide 2; ▾, peptide 3. Fields virology.
(A) The average objective function value in 16 drug combinations reduced as iteration moves on. Philadelphia, PA: Lippincott Williams & Wilkins; 2007. Notably, the reaction containing 100 copies of HIV-1 DNA that was classified as negative produced a faintly visible line at the test zone of the lateral flow strip (Fig. 2501–2601.