Functional exchangeability of the nuclear localization signal (NLS) of capsid protein between PCV1 and PCV2 in vitro: Implications for the role of NLS in viral replication

<p>Abstract</p> <p>Background</p> <p>Porcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication.</p> <p>Results</p> <p>The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 10^4.2and 10^3.8TCID₅₀/ml, which were significantly lower than that of PCV1 and PCV2 (10^5.6and 10^5.0TCID₅₀/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2.</p> <p>Conclusions</p> <p>Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.</p

Back-stepping control based on extended state observer for magnetic levitation ball

Aiming at the characteristics of non-linearity,uncertainty and susceptibility to disturbance of single-degree-of-freedom magnetic levitation ball system,a back-stepping control method based on extended state observer (ESO-BS) was proposed to improve the control performance of the system.The extended state observer was used to estimate the position,velocity and disturbance information of the levitation ball in real time when the system was disturbed by uncertainty.The estimation was combined with the controller design,then the back-stepping method was used to design the levitation position tracking control law of the magnetic levitation ball,and the Lyapunov method was used to prove the ultimate bounded convergence of the tracking error of the system.The simulation results show that,compared with PID control,the adjusting time of ESO-BS control system is 0.01 s,while that of PID control is 0.08 s,which is obviously longer,so the dynamic characteristic of ESO-BS control is better than that of PID control.When there is uncertainty in the system,the designed control law can realize the stable suspension of the ball,and can realize the position tracking according to the required suspension height position

Effects of Different Oxytocin and Temperature on Reproductive Activity in Nile tilapia (Oreochromis niloticus): Based on Sex Steroid Hormone and GtHR Gene Expression

Luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) belong to the gonadotropic hormone receptors (GtHR), which are highly expressed in fish gonads and participate in the regulation of fish reproductive activities. Fish gonadal development and gamete maturation are not only regulated by their BPG axis but also affected by natural environmental factors (such as temperature, salinity, pH, nutrients, light, etc.). Nile tilapia (Oreochromis niloticus) is a farmed fish with a short reproductive cycle, fast growth, and high economic value. To study the relationship between gonadotropic hormone receptors (GtHR) and the reproductive activity of Nile tilapia, different oxytocin injection experiments and different temperature treatment experiments were set up, and the expression changes of the GtHR gene in the gonads and the concentration changes of the estradiol (E2) in the female serum and testosterone (T) in the male serum were determined employing a quantitative RT-PCR assay and enzyme-linked immunosorbent assay (ELISA), respectively. After the injection of oxytocin, with the change of E2 in females and T in males, the FSHR showed an expression pattern of first increase, then decrease, and the LHR showed an expression pattern of first increase, then decrease, and finally increase in the gonads, and the expression level of FSHR and LHR in the injection group was significantly higher than that in the control group at multiple time points; in addition, the expression level of FSHR and LHR in the oxytocin-combination injection group was higher than that in the single injection group. During 28 days of treatment at different temperatures, the sex steroid hormones and GtHR genes also showed regular changes, and the relationship between each group was 28 &deg;C &gt; 32 &deg;C &gt; 24 &deg;C at most time points. According to the research results, it is speculated that FSHR and LHR play an important role in the development of Nile tilapia gonads and participate in the reproductive activities of Nile tilapia. By comparing and analyzing the changes in the sex steroid hormones and GtHR genes in each experimental group, it is speculated that different oxytocin injections could affect the expression of FSHR and LHR genes in Nile tilapia, and the combined effect of oxytocin was better than single oxytocin; the optimum temperature for the reproduction of Nile tilapia is between 28&ndash;32 &deg;C. This study provides a theoretical basis for further elucidating the physiological functions and molecular mechanisms of FSHR and LHR and also provides a reference for the research of reproductive regulation in Nile tilapia

Transcriptome Profiling Revealed Basis for Growth Heterosis in Hybrid Tilapia (<i>Oreochromis niloticus</i> ♀ × <i>O. aureus</i> ♂)

Hybrid tilapia were produced from hybridization of Nile tilapia (Oreochromis niloticus) and blue tilapia (O. aureus). Comparative transcriptome analysis was carried out on the liver of hybrid tilapia and their parents by RNA sequencing. A total of 2319 differentially expressed genes (DEGs) were identified. Trend co-expression analysis showed that non-additive gene expression accounted for 67.1% of all DEGs. Gene Ontology and KEGG enrichment analyses classified the respective DEGs. Gene functional enrichment analysis indicated that most up-regulated genes, such as FASN, ACSL1, ACSL3, ACSL6, ACACA, ELOVL6, G6PD, ENO1, GATM, and ME3, were involved in metabolism, including fatty acid biosynthesis, unsaturated fatty acid biosynthesis, glycolysis, pentose phosphate pathway, amino acid metabolism, pyruvate metabolism, and the tricarboxylic acid cycle. The expression levels of a gene related to ribosomal biosynthesis in eukaryotes, GSH-Px, and those associated with heat shock proteins (HSPs), such as HSPA5 and HSP70, were significantly down-regulated compared with the parent tilapia lineages. The results revealed that the metabolic pathway in hybrid tilapia was up-regulated, with significantly improved fatty acid metabolism and carbon metabolism, whereas ribosome biosynthesis in eukaryotes and basal defense response were significantly down-regulated. These findings provide new insights into our understanding of growth heterosis in hybrid tilapia

Integration of mRNA and miRNA Profiling Reveals Heterosis in <i>Oreochromis niloticus</i> × <i>O. aureus</i> Hybrid Tilapia

Heterosis is a widespread biological phenomenon in fishes, in which hybrids have superior traits to parents. However, the underlying molecular basis for heterosis remains uncertain. Heterosis in growth and survival rates is apparent in hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂). Comparisons of growth and hematological biochemical characteristics and mRNA and miRNA transcriptional analyses were performed in hybrid and parents tilapia stocks to investigate the underlying molecular basis for heterosis. Growth characteristics and hematological glucose and cholesterol parameters were significantly improved in hybrids. Of 3097 differentially expressed genes (DEGs) and 120 differentially expressed miRNAs (DEMs) identified among three stocks (O. niloticus, O. aureus, and hybrids), 1598 DEGs and 62 DEMs were non-additively expressed in hybrids. Both expression level dominance and overdominance patterns occurred for DEGs and DEMs, indicating that dominance and overdominance models are widespread in the transcriptional and post-transcriptional regulation of genes involved in growth, metabolism, immunity, and antioxidant capacity in hybrid tilapia. Moreover, potential negative regulation networks between DEMs and predicted target DEGs revealed that most DEGs from miRNA-mRNA pairs are up-regulated. Dominance and overdominance models in levels of transcriptome and miRNAome facilitate the integration of advantageous parental alleles into hybrids, contributing to heterosis of growth and improved survival. The present study provides new insights into molecular heterosis in hybrid tilapia, advancing our understanding of the complex mechanisms involved in this phenomenon in aquatic animals