Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alterations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homologous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1% in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G 1 /S-phase cells in the target population through the addition of thymidine moderately (ϳ2-fold) increased gene correction compared to cells in other cell cycle phases, including G 0 /G1, G 1 , and G 2 /M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAAV targeting efficiencies in primary fetal fibroblast and tibialis muscles. However, targeting efficiencies in primary mouse fetal fibroblasts were significantly lower (ϳ0.006%) than in 293 cells, and no correction was seen in tibialis muscles following rAAV infection. To evaluate the molecular structures of rAAV genomes that might be responsible for gene repair, single-cell injection studies were performed with purified viral DNA in a mutant eGFP target cell line. However, the failure of direct cytoplasm-or nucleus-injected rAAV DNA to facilitate gene repair suggests that some aspect of intracellular viral processing may be required to prime recombinant viral genomes for gene repair events.
Viral protein 2 (VP2) of porcine parvovirus (PPV) is the major viral structural protein and is responsible for eliciting neutralizing antibodies in immunized animals. In this study, we constructed and characterized a recombinant yeast vector encoding the VP2 protein, designated as pGAPZαA-VP2. The construct was confirmed by restriction enzyme digestion, PCR, and sequencing and then introduced into P. pastoris strain SMD1168 by electroporation. The expressed VP2 protein was analyzed by SDS-PAGE and western blot. Immunization of mice with the VP2 protein elicited a PPV-specific humoral immune response. Notably, a preparation of VP2 protein containing adjuvant induced a much better antibody response than VP2 alone. Clearly, the adjuvant strongly enhanced the immunogenicity of VP2. This study provides a foundation for the application of the VP2 protein in the clinical diagnosis of PPV and in vaccination against PPV in the future.
BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccines have specific limitations in terms of their safety and efficacy, so the development of novel antiviral drugs is urgently required. The aim of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of the antimicrobial peptide cecropin P1 (CP1) against PRRSV infection in vitro.ResultsCP1 not only displayed extracellular virucidal activity against PRRSV, but also exerted a potent inhibitory effect when added either before, simultaneously with, or after viral inoculation. The inhibitory effect of CP1 occurred during viral attachment, but not at viral entry into Marc-145 cells. CP1 also inhibited viral particle release and attenuated virus-induced apoptosis during the late phase of infection. CP1 exerted similar inhibitory effects against PRRSV infection in porcine alveolar macrophages, the cells targeted by the virus in vivo during its infection of pigs. The expression of interleukin 6 was elevated by CP1 in porcine alveolar macrophages, which might contribute to its inhibition of PRRSV infection.ConclusionsCollectively, our findings provide a new direction for the development of potential therapeutic drugs against PRRSV infection.
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