Multiple-step Growth Research Articles

One-Step Assembly of a PRRSV Infectious cDNA Clone and a Convenient CRISPR/Cas9-Based Gene-Editing Technology for Manipulation of PRRSV Genome.

Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.

Analysis on the dislocation of GaN on different substrate

Though there are still disputes on whether the threading dislocation density (TDD), to produce high quality Gallium Nitride ( GaN ) with low TDD is needed. In this passage, we would first show the impact of dislocation and review the latest methods to reduce dislocation on Si, GaN, and sapphire. The Si substrating with serpentine channels could produce GaN by using Si substrate with low density dislocations. The method that use the markless-3D provides a new way to reduce the dislocation from big off-angle. The multiple-step growth technique controls the conditions of crystallization and use two steps of crystallizing to grow with both low dislocation density and 2D growth shape. The last method is to use the Na-flux method to exclude cracks with sapphire in crystal.

Phase Control of Ga<sub>2</sub>O<sub>3</sub> Thin Films Grown by Metal-Organic Chemical Vapor Deposition

In this paper, Ga2O3 thin films were grown on c-plane sapphire substrates by metal-organic chemical vapor deposition (MOCVD). There was phase transition for samples grown with different flow rates of triethyl-gallium (TEGa) and deionized water (H2O). It is found that ε-Ga2O3 is difficult to coalesce and the phase mixture by β­Ga2O3 takes place if the flow rates of TEGa and H2O are too high. However, by using multiple-step growth method, the film became fully coalesced. High-quality ε-Ga2O3 thin film with atomically flat surface and multilayer morphology was obtained.

Bulk GaN substrate with overall dislocation density on the order of 105/cm2 fabricated by hydride vapor phase epitaxy

In this study, a combined facet and flattening (FF) growth technique was implemented to fabricate GaN substrates by hydride vapor phase epitaxy. By changing the growth conditions, i.e., the growth temperature and V/III ratio, it was found that facet growth was promoted with a high V/III ratio and low temperature and planar growth was promoted with low V/III ratios and high temperature. We introduce a FF growth technique involving further reduction of the dislocation density using facet growth as the first step and flattening growth of the GaN layer as the second step. To further reduce dislocation density, we also finally demonstrate a multiple-step growth technique based on FF growth and succeeded in producing GaN substrates with overall dislocation densities on the order of 105cm−2.

Rana grylio virus TK and DUT gene locus could be simultaneously used for foreign gene expression

Ranaviruses (family Iridoviridae, genus Ranavirus) have been recognized as emerging infectious pathogens and caused a great loss to the global biodiversity. Thymidine kinase (TK) and deoxyuridine triphosphatase (dUTPase, DUT, encoded by ORF 67R) are ubiquitous, existing in iridoviruses and other organisms. Previous studies showed that TK and DUT could be individually knocked out without impeding viral replication. In this study, we tried to insert two fluorescence genes into the above loci. We started with Δ67R-RGV, a recently generated recombinant Rana grylio virus (RGV) with the whole DUT replaced by enhanced green fluorescence protein (EGFP) gene. Then, a red fluorescence protein (RFP) gene initiated by RGV immediate-early (IE) ICP18 gene promoter was inserted into TK locus through homologous recombination. A novel recombinant virus, ΔDUT, TK-RGV, was generated by nine successive rounds of plaque isolation using RFP selection. All of the plaques produced by this recombinant virus could emit both green and red fluorescence. Furthermore, one-step and multiple-step growth curves of ΔDUT, TK-RGV were similar to those of wt-RGV and Δ67R-RGV. In conclusion, a novel dual-fluorescence labeled recombinant iridovirus in which DUT and TK gene locus were simultaneously used for foreign gene expression was constructed.

Gene order rearrangement of the M gene in the rabies virus leads to slower replication.

The matrix protein (M) is one of only five genes in the RV genome and is an important multifunctional protein. Besides to allow for the release of newly replicated virions pairing with G, the M protein also functions in virus replication, pathogenicity, and host cell apoptosis. The goal of present study is to generate recombinant viruses with M gene rearranged, thus laying the foundation for further exploring what will happen when the gene for M is relocated on the RV single-strand RNA. We used rHEP-Flury, an attenuated virus that remains virulent for less than 3days in sucking mice, to reshuffle the M gene, using an approach that leaves the other viral nucleotide sequence intact. Two viruses with translocated M genes (N1M2 and N1M4) were recovered from each of the rearranged cDNAs, whose gene order is 3'-N-M-P-G-L-5' and 3'-N-P-G-M-L-5' respectively. The growth dynamics of these viruses showed slower replication than the wild-type virus in multiple-step growth curves, but they can grow to a comparable titer in tests of single-step growth curves. Further experimentation with these rearranged viruses will provide insights into the relationships between genome structure and virus phenotypes.

Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus.

Multiple Step Growth of Single Crystalline Rutile Nanorods with the Assistance of Self-Assembled Monolayer for Dye Sensitized Solar Cells

A novel multiple step growth (MSG) process has been developed to synthesize rutile nanorods (NRs) on fluorine-doped tin oxide (FTO) glass with the assistance of a self-assembled monolayer (SAM) aiming to increase the internal surface area of the 1D materials for dye sensitized solar cell (DSSC) applications. The experimental result reveals that the SAM layer can be selectively decomposed at the tip of the nanorod, namely the rutile (001) surface, due to the anisotropic photocatalytic property of the rutile. The remaining SAM layer on the side-wall of the NRs remains intact and serves as water repellent which prevents the radial growth of the NRs during the next step hydrothermal synthesis; therefore, the spacing between the NRs and the porosity of the NR array can be retained after additional growth cycles. On the other hand, introduction of a middle layer formed via TiCl4 solution treatment before the next growth cycle is found to be an effective way to control the diameters of the newly grown NRs. The performance of DSSC made from the rutile NRs grown using the MSG technique has been examined, and it is significantly affected by the internal surfaces of the NRs. Furthermore, the MSG combined with NR etching treatment by acid at low temperature (150 °C) leads to a significant enhancement in the solar cell performance. The gigantic wettability difference of the NRs before and after the SAM treatment as well as the MSG method could be adapted to prepare superhydrophobic and superhydrophilic nanostructured patterns for other applications.

Joint production of prime/boost pairs of Fowlpox Virus and Modified Vaccinia Ankara recombinants carrying the same transgene

Pairs of recombinant MVA (Modified Vaccinia Ankara) and FPV (Fowlpox Virus) expressing the same transgene are reasonable candidates for prime/boost regimens, because cross-reacting immune responses between the two vectors, both non-replicative in mammalian hosts, are very limited. The acceptor virus FPD-Red, a derivative of FPV, carrying a red fluorescent protein gene flanked by the homology regions of MVA deletion III, was constructed. The same MVA Transfer Plasmid Green, designed to insert transgenes into the MVA deletion III locus, can therefore be used to transfer transgenes into both acceptor viruses MVA-Red and FPD-Red with the described recently Red-to-Green gene swapping method. Cells infected by either recombinant virus can be sorted differentially by a simple and reliable FACS-based purification protocol. The procedure is carried out in primary chick embryo fibroblasts grown in serum-free media and was applied to the production of three rMVA/rFPV pairs expressing the H5N1 avian influenza antigens M1, M2 and NP. The viral genes were human codon-optimized and expressed at high levels in both chick and mammalian cells. Both single-step and multiple-step growth analyses showed no significant differences in growth due to the transgenes in either rMVA or rFPV derivatives.