Cowpea mosaic virus-based chimaeras. Effects of inserted peptides on the phenotype,host range,and transmissibility of the modified viruses

Expression of foreign peptides on the surface of cowpea mosaic virus particles leads to the creation of chimaeras with a variety of phenotypes and yields. Two factors were shown to be particularly significant in determining the properties of a given chimaera: the length of the inserted sequence and its isoelectric point. The deleterious effect of high isoelectric point on the ability of chimeras to produce a systemic infection occurs irrespective of the site of insertion of the peptide. Ultrastructural analysis of tissue infected with chimaeras with different phenotypes showed that all produced particles with a tendency to aggregate, irrespective of the size or isoelectric point of the insert. Host range and transmission studies revealed that the expression of a foreign peptide did not (1) alter the virus host range, (2) increase the rate of transmission by beetles or through seed, or (3) change the insect vector specificity. These findings have implications for both the utility and the biosafety of Cowpea mosaic virus-based chimaeras.     

A rapid, efficient and economical method for generating leishmanial gene targeting constructs

Targeted gene replacement is a powerful tool in Leishmania genetics that can be time-consuming to implement. One tedious aspect that delays progress is the multi-step construction of gene targeting vectors. To accelerate this process, we developed a streamlined method that allows the assembly of a complete targeting vector from all its constituent parts in a single-step multi-fragment ligation. The individual components to be assembled are flanked by sites for the restriction endonuclease SfiI that generates non-identical, non-palindromic three base 3'-overhangs designed to allow annealing and ligation of the parts only in the proper order. The method was optimized by generating constructs for targeting the Leishmania donovani inosine monophosphate dehydrogenase gene (LdIMPDH) encoding six different drug resistance markers, and was found to be rapid and efficient. These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species.     

Development of cowpea mosaic virus-based vectors for the production of vaccines in plants

Plant viruses are emerging as an attractive alternative to stable genetic transformation for the expression of foreign proteins in plants. The main advantages of using this strategy are that viral genomes are small and easy to manipulate, infection of plants with modified viruses is simpler and quicker than the regeneration of stably transformed plants and the sequence inserted into a virus vector will be highly amplified. One use of these virus expression systems is for vaccine production. Among plant viruses, cowpea mosaic virus makes an ideal candidate for the production of such vaccines because it grows extremely well in host plants, is very stable, and the purification of virus particles, if required, is straightforward. In this article, the authors review the progress made in the development of cowpea mosaic virus-based vectors for vaccine production, making use of two main approaches: epitope presentation and polypeptide expression.     

Development of a new cucumber mosaic virus-based plant expression vector with truncated 3a movement protein

We have developed a Cucumber mosaic virus (CMV)-based expression vector for the production of heterologous proteins in plants. Cell-to-cell movement of CMV is dependent on the presence of coat protein (CP). Previous studies have shown that deletion of 33 amino acids (aa) from the carboxy-terminus of the 3a movement protein facilitates cell-to-cell movement that is independent of CP. The CMV-based expression vector that we have designed utilizes this truncated 3a protein, allowing the expression of target genes from the strong CP subgenomic promoter and without the need for providing CP in trans for cell-to-cell spread. Using this vector we achieved expression levels of ~ 450 mg/kg leaf tissue of green fluorescent protein (GFP) when the vector was delivered into Nicotiana benthamiana plants by agroinfiltration. Human growth hormone (hGH), on the other hand, accumulated to ~ 170 mg/kg of leaf tissue when the same approach was used to deliver the vector.     

Vitrification as a prospect for cryopreservation of tissue-engineered constructs

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation.     

Systemic gene delivery into soybean by simple rub-inoculation with plasmid DNA of a Soybean mosaic virus-based vector

Archives of Virology -     

Assessment of a bovine co-culture, scaffold-free method for growing meniscus-shaped constructs

Using a self-assembly (SA), scaffoldless method, five high-density co-cultures with varied ratios of meniscal fibrochondrocytes (MFCs) and articular chondrocytes (ACs) were seeded into novel meniscus-specific, ring-shaped agarose wells. The following ratios of MFCs to ACs were used: 0% MFC, 25% MFC, 50% MFC, 75% MFC, and 100% MFC. Over 4 weeks, all ratios of cells self-assembled into three-dimensional constructs with varying mechanobiological and morphological properties. All groups stained for collagen II (Col II), and all groups except the 0% MFC group stained for collagen I (Col I). It was found that the tensile modulus was proportional to the percentage of MFCs employed. The 100% MFC group yielded the greatest mechanical stiffness with 432.2 +/- 47 kPa tensile modulus and an ultimate tensile strength of 23.7 +/- 2.4 kPa. On gross inspection, the 50% MFC constructs were the most similar to our idealized meniscus shape, our primary criterion. A second experiment was performed to examine the anisotropy of constructs as well as to directly compare the scaffoldless, SA method with a poly-glycolic acid (PGA) scaffold-based construct. When compared to PGA constructs, the SA groups were 2-4 times stiffer and stronger in tension. Further, at 8 weeks, SA groups exhibited circumferential fiber bundles similar to native tissue. When pulled in the circumferential direction, the SA group had significantly higher tensile modulus (226 +/- 76 kPa) than when pulled in the radial direction (67 +/- 32 kPa). The PGA constructs had neither a directional collagen fiber orientation nor differences in mechanical properties in the radial or circumferential direction. It is suggested that the geometric constraint imposed by the ring-shaped, nonadhesive mold guides collagen fibril directionality and, thus, alters mechanical properties. Co-culturing ACs and MFCs in this manner appears to be a promising new method for tissue engineering fibrocartilaginous tissues exhibiting a spectrum of mechanical and biomechanical properties.     

Evaluation of E test as a rapid method for determining MICs for nutritionally variant streptococci.

E test was evaluated as an alternative rapid and simple method of MIC estimation for nutritionally variant streptococci. E test with various media was compared with conventional broth and plate dilution techniques supplemented with 0.001% (wt/vol) pyridoxal hydrochloride (vitamin B6). Of the 14 strains tested with E test, isosensitest agar supplemented with 5% defibrinated horse blood and 0.001% pyridoxal HCl, with and without 0.01% cysteine, gave complete agreement within one twofold-dilution titer of the agar reference method and between 93 and 86% agreement within one twofold-dilution titer of the broth reference method. E test MICs with other media were comparable; however, these were considerably more difficult to interpret. Use of Mueller-Hinton and Columbia-based supplemented agar showed hazy growth and double zoning around the endpoint, respectively. The addition of 0.01% (wt/vol) cysteine to media exhibited no significant effect, and incubation in 5% carbon dioxide (CO2) did not affect MICs.     

Half-genome human immunodeficiency virus type 1 constructs for rapid production of reporter viruses

The use of reporter constructs of HIV-1 molecular clones has been useful in studying the effects of infection on individual cells. A strategy of producing viral half-genome constructs containing reporter genes that can be used to produce recombinant complete infectious virions is described. This approach allows rapid generation of reporter and non-reporter virions from the same genetic construct, obviating the need to produce a new reporter genetic construct for each viral change to be studied. We provide an example of the utility of this system for studying the effects of Nef on infected cells.     

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.     

Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector

A major obstacle in the genetic manipulation of tomato mosaic virus (ToMV) is the instability of the plasmid containing the infectious full-length cDNA of the ToMV vector, which often prevents the subcloning of a foreign gene of interest into the vector. We found that an insertion of a 0.3-1.6-kbp DNA fragment in the movement protein (MP) coding region effectively attenuated bacterial toxicity of the plasmid and greatly increased plasmid yield. Accumulation of a modified ToMV containing a 0.3-kb insertion in the MP coding region was comparable to that of a modified ToMV without an insertion in tobacco BY-2 protoplasts, while an insertion more than 0.6 kb significantly reduced accumulation of the viral RNA. The modified ToMV vector containing a 0.3-kb insertion was easily manipulated to introduce a coding sequence for human interferon-gamma (HuIFN-gamma) and successfully utilized to produce HuIFN-gamma in both BY-2 protoplasts and transgenic BY-2 cells.     

A rapid seeding technique for the assembly of large cell/scaffold composite constructs

These studies address critical technical issues involved in creating human mesenchymal stem cell (hMSC)/ scaffold implants for cartilage repair. These issues include obtaining a high cell density and uniform spatial cell distribution within the scaffold, factors that are critical in the initiation and homogeneity of chondrogenic differentiation. For any given scaffold, the initial seeding influences cell density, retention, and spatial distribution within the scaffold, which eventually will affect the function of the construct. Here, we discuss the development of a vacuum-aided seeding technique for HYAFF -11 sponges which we compared to passive infiltration. Our results show that, under the conditions tested, hMSCs were quantitatively and homogeneously loaded into the scaffolds with 90+% retention rates after 24 h in perfusion culture with no negative effect on cell viability or chondrogenic potential. The retention rates of the vacuum-seeded constructs were at least 2 times greater than those of passively seeded constructs at 72 h. Histomorphometric analysis revealed that the core of the vacuum-seeded constructs contained 240% more cells than the core of passively infiltrated scaffolds. The vacuum seeding technique is safe, rapid, reproducible, and results in controlled quantitative cell loading, high retention, and uniform distribution.     

A procedure for rapid isolation and analysis of cauliflower mosaic virus DNA

A simple and rapid procedure has been developed for the small-scale isolation of crude preparations of cauliflower mosaic virus DNA. The preparations are relatively free of contaminating cellular DNA and are sufficiently pure to be digested with restriction enzymes. The method enables plants infected with single-lesion isolates of mutagenized viral DNA to be rapidly screened for particular restriction endonuclease sites.     

Evaluation of a rapid method for identifying Branhamella catarrhalis.

The speed and precision of hydrolysis of tributyrin to butyric acid as a test to detect Branhamella catarrhalis were evaluated. The test proved consistently reliable in the identification of strains and correctly differentiated B catarrhalis from Neisseria sp. The combination of Gram stain, oxidase, and catalase tests to tributyrin hydrolysis provides a means of positive same day identification of B catarrhalis.