A novel single-cell quantitative real-time RT-PCR method for quantifying foot-and-mouth disease viral RNA.

Foot-and-mouth disease virus is a positive-sense, single-stranded RNA virus with a negative strand as its replication intermediate, which can cause severe acute infection in sensitive cell lines. To investigate better the actual state of virus infection, there is a need to measure the amount of FMDV RNA in a single acutely infected cell rather than in a large number of cells. Therefore, in the present study, a strand-specific single-cell quantitative real-time RT-PCR was developed to analyze the RNA or FMDV. This new method uses two techniques in concert with each other: a technique for isolating single cells with micromanipulators, which is coupled to an assay for detecting viral RNA by real-time RT-PCR. In the assay of acute infection, 185 of 224 (82.6%) single-cell samples were positive and contained viral genome copies ranging from several to thousands, and up to 1,000,000 copies. However, not all cells were infected and there were differences in the number of viral RNA copies between cells. A single-cell quantitative RT-PCR was validated to be feasible and effective.

A negative correlation between water content and protein adsorption on polymer brushes.

The correlation between the water content and protein adsorption on the surface of polymer brushes is investigated quantitatively. Using a number of model systems such as poly N-isopropylacrylamide (PNIPAM), polyethylene glycol (PEG), poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(2,2,3,4,4,4-hexafluorobutyl)methacrylate (PHFBMA) polymer brushes with various grafting densities, the amount of water hydrodynamically coupled inside the brushes and its correlation with protein adsorption (BSA and Fg proteins as model systems) were determined by quartz crystal microbalance with dissipation (QCM-D) and surface plasma resonance (SPR). The results demonstrate the negative correlation between the water content and protein adsorption - the more water is coupled inside the brushes, the more protein adsorption is suppressed. In particular for PNIPAM brushes with a high enough grafting density and with a water content greater than 250 ng cm-2, the protein adsorption is negligible.

Biocompatible magnetite nanoparticles synthesized by one-pot reaction with a cell membrane mimetic copolymer.

In this paper, a series of random copolymers poly(methacrylic acid -co-2-methacryloyloxyethyl phosphorylcholine) P(MAA-co-MPC) were synthesized firstly via RAFT living polymerization. The P(MAA-co-MPC) copolymer side chains bear cell membrane phosphorylcholine zwitterions to endow biocompatibility and carboxylic groups to confer coordination with metal ions. Thus, the copolymer was adopted to modify Fe3O4 nanoparticle by a one-pot coprecipitation approach. The effects of the copolymer composition as well as the ratio between the copolymers and iron ions on the performances of the magnetite nanoparticles were researched. The diameters of the nanoparticles could be easily tuned by changing the initial copolymer amount. Moreover, a long-term colloidal stability of magnetite particles was obtained after P(MAA-co-MPC) modification. Biocompatibility of the P(MAA-co-MPC) copolymer coated magnetite nanoparticles was investigated by protein adsorption, in vitro cytotoxicity and cell uptake studies. It was found that the copolymer content of magnetite nanoparticles correlates with its biocompatibility. Excellent biocompatibility could be obtained when the content of the copolymer in the composite nanoparticles reached to 54%.

An antiviral mechanism investigated with ribavirin as an RNA virus mutagen for foot-and-mouth disease virus.

To prove whether error catastrophe/lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of Rp1 to Rp5 were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of Rp1 to Rp5 and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from Rp1 to Rp5, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the Rp5-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of 1000 microM and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutagenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.