Search

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to be highly heterogeneous and variable among PRRSV strains and some sequences in the middle region of the nsp2 are not essential to viral replication. Recent studies have attempted to insert foreign genes in the nsp2 nonessential regions but the foreign genes were not stably expressed by recombinant viruses in vitro. In the present study, we first constructed an infectious cDNA clone with deletion of 75 nucleotides (25 amino acids) in the nsp2 region (rHuN4-F112-Δ508-532) of the attenuated vaccine virus HuN4-F112 derived from a highly pathogenic PRRSV HuN4 and then inserted a gene fragment encoding a immunodominant B-cell epitope (49 amino acids) of Newcastle disease virus (NDV) nucleoprotein (NP) in-frame into the deletion site. The viable recombinant virus was rescued from the full-length cDNA infectious clone in vitro. The engineered viruses rescued from the cDNA clone indicated that the deletions of 75 nucleotides and insertion of NDV NP gene in the nsp2 region did not affect viral replication; they had similar growth kinetics to its parental virus. The inserting gene could be expressed consistently when the recombinant virus was passaged up to twenty times in cell cultures as determined by immunofluorescence assay (IFA) and genomic sequencing. To investigate the potential application of the NDV NP gene-inserted PRRSV as a marker vaccine, piglets were immunized with the recombinant virus and then challenged with lethal dose of highly pathogenic PRRSV. The immunized piglets produced specific antibodies against both the NDV NP and PRRSV, and lacked antibodies against the deleted 25aa nsp2 epitope. After challenge, all immunized piglets were protected from clinical disease or death, while all piglets in control group died (5/5) by ten days post challenge. The results of the present study indicated that the recombinant PRRSV (rHuN4-F112-Δ508-532) could be used as a potential marker vaccine against PRRS.

As the complexity of design and fabrication for instrumentation-on-silicon systems increases, the optimization of a combined Built-In Self-Test (BIST) and Automated Test Equipment (ATE) process is desirable to meet the high fault coverage while preserving acceptable costs. For digital systems, the costs associated with a combined BIST/ATE testing process mainly consist of the following components: 1) the cost due to the BIST area overhead and 2) the cost due to the overall test time. In general, BIST is faster than ATE, but it can provide only a limited fault coverage; for attaining a higher fault coverage from BIST, additional area (at a corresponding higher cost) is required. However, a higher fault coverage can usually be achieved from ATE, but excessive use of ATE results in additional test time (as an increased cost). The fault coverage of BIST and ATE plays a significant role, because it can affect the area overhead in BIST and the test time in BIST/ATE. This paper proposes a novel numerical method to find the optimized fault coverage by BIST and ATE so that minimal cost can be achieved. The proposed method is then applied to two parallel combined BIST/ATE testing schemes to ensure its validity

Using mutation PCR, we cloned the target gene containing 421-480nt (141-160aa) and 598-639nt (200-213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508-532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by transfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50 = -log10(-6.75)/0.1 mL) was similar to its direct parental virus rHuN4-F112-delta508-532, but higher than rHuN4-F112.

Background Nowadays, PRRS has become one of the most economically important infectious diseases of pig worldwide. To better characterize and understand the molecular basis of PRRSV virulence determinants, it would be important to develop the infectious cDNA clones. In this regard, HuN4-F112, a live-attenuated North-American-type PRRSV vaccine strain, could serve as an excellent model. Results In the study, genomic sequence of HuN4-F112, an attenuated vaccine virus derived from the highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) HuN4 strain, was determined and its full-length cDNA was cloned. Capped RNA was transcribed in vitro from the cDNA clone and transfected into BHK-21 cells. The supernatant from transfected monolayers were serially passaged in Marc-145 cells. The rescued virus exhibited a similar growth pattern to its parental virus in Marc-145 cells with peak titers at 48 h post-infection. Conclusion In conclusion, we rescued virus from an infectious cDNA clone of attenuated vaccine. It is possible in the future that a new attenuated PRRSV vaccine with broader specificity and good immunogenicity can be designed in vitro via an infectious cDNA clone platform coupled with validated information on virulence determinants.

The development of cell-mediated immunity has been known extremely important in clearing porcine reproductive and respiratory syndrome virus (PRRSV) in infected pigs. However, the PRRS immunology regarding the interaction of T-cells and PRRSV proteins is poorly understood. To identify the T-cell immunodominant epitopes on the membrane (M) protein of PRRSV, a series of 31 overlapping pentadecapeptides covering the entire M protein were designed and synthesized. These peptides were screened by ELIspot analysis for their capabilities to elicit interferon-gamma (IFN-γ) responses in the peripheral blood mononuclear cells (PBMCs), which were collected from pigs immunized with attenuated PRRSV HuN4-F112 strain and challenged with highly pathogenic HuN4 strain. After three rounds of screening, 4 peptides (M3, M6, M8 and M12) were shown to elicit high expression of IFN-γ. The stimulation of high IFN-γ transcription in PBMCs by these 4 peptides was further confirmed in real-time PCR. The sequence alignment revealed that the epitope represented by peptide M6 was fully conserved in all of examined 42 North American genotype II PRRSV isolates and the epitopes represented by peptides M3, M8 and M12 showed 2-4 amino acid replacements. The finding of 4 T-cell immunodominant epitopes in the M protein of PRRSV will be beneficial to the understanding of the development of cell-mediated immunity.

The recent development of nanoscale materials and assembly techniques has resulting in the manufacturing of high-density computational systems. These systems consist of nanometer-scale elements and are likely to have many manufacturing imperfections (defects); thus, defect-tolerance is considered as one of the most some algorithms for repairing defective crosspoints in a nanoscale crossbar architecture; furthermore we estimate the efficiency and cost-effectiveness of each algorithm. Also, for a given design and manufacturing environment, we propose a cost-driven method to find a balanced solution by which figures of merit such as area, repair time and reconfiguration cost can be taken into account. Probabilistic parameters are utilized in the proposed cost-driven method for added flexibility.

Recent developments for biosensors have been mainly focused on miniaturization and exploratory use of new materials. It should be emphasized that the absence of a novel "in-situ self-calibration/diagnosis technique" that is not connected to an external apparatus is a key obstacle to the realization of a biosensor for continuous use with minimum attendance. In order to address this issue, a novel solid-state glucose oxidase-coupled amperometric biosensor with integrated electrochemical actuation system has been designed and validated. There are two key components of the proposed glucose biosensor: solid-state GOD-coupled thin-1m amperometric sensing element and O2 depleting/saturating built-in electrochemical actuator. The actuator can be used to accomplish in-situ 1-point self-calibration by depleting O2 (i.e., by simulating glucose-free environment). Also, it can be used at the same time to extend the proposed sensor's linear detection range by increasing O2 (i.e., by enhancing glucose sensitivity). A prototype sensor was fabricated and a series of lab experiments was conducted. Collected data assures that the proposed sensor effectively establishes the zero calibration point and significantly enhances its measurement sensitivity and confidence

With molecular-scale materials and fabrication techniques recently developed, high-density computing systems in nanometer domain emerge. An array-based nanoarchitecture has been recently proposed based on nanowires such as carbon nanotubes (CNTs), silicon nanowires (SiNWs). High-density nanoarray-based systems consisting of nanometer-scale elements are likely to have many imperfections; thus, defect-tolerance is considered as one of the most significant challenges. In this paper, we propose a probabilistic yield model for the array-based nanoarchitecture. The proposed yield model can be used 1) to accurately estimate the raw and net array densities, and 2) to design and optimize more defect and fault-tolerant systems based on the array-based nanoarchitecture.

As the design and fabrication complexities for the instrumentation-on-silicon systems intensify, optimization of combined Built-In Self-Test (BIST) and Automated Test Equipment (ATE) testing becomes more desirable to meet the required fault-coverage while maintaining acceptable cost overhead. The cost associated with combined BIST/ATE testing of such systems mainly consists of the following; (1) the cost induced by the BIST area overhead and (2) the cost induced by the overall testing time. In general, BIST has faster testing speed than ATE, while it can provide only limited fault-coverage and driving higher fault-coverage from BIST means additional area cost overhead. On the other hand, higher fault-coverage can be usually achieved from ATE, but excessive use of ATE results in additional test time cost. Fault-coverage of BIST and ATE plays a significant role since it can affect the area overhead in BIST and test time in BIST/ATE. This paper is to propose a novel numerical method to find an optimized fault-coverage implemented in BIST and ATE so that a minimum cost can be achieved. The proposed method. then, is applied to two parallel combined BIST/ATE testing schemes to assure its technical validity.

With molecular-scale materials, devices and fabrication techniques recently being developed, high-density computing systems in the nanometer domain emerge. An array-based nanoarchitecture has been recently proposed based on nanowires such as carbon nanotubes (CNTs) and silicon nanowires (SiNWs). High-density nanoarray-based systems consisting of nanometer-scale elements are likely to have many imperfections; thus, defect-tolerance is considered one of the most significant challenges. In this paper we propose a probabilistic yield model for the array-based nanoarchitecture. The proposed yield model can be used (1) to accurately estimate the raw and net array densities, and (2) to design and optimize more defect and fault-tolerant systems based on the array-based nanoarchitecture. As a case study, the proposed yield model is applied to the defect-tolerant addressing scheme called h-hot addressing and simulation results are discussed.

Background The regions in the middle of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) have been shown to be nonessential for PRRSV replication, and these nonessential regions are different in various viral strains. Finding In this study, the nonessential regions of the nsp2 of an attenuated vaccine strain (HuN4-F112) of highly pathogenic porcine reproductive and respiratory syndrome virus were identified based on an infectious cDNA clone of HuN4-F112. The results demonstrated that the segments of nsp2 [amino acids (aa) 480 to 667] tolerated deletions. Characterization of the mutants demonstrated that those with small deletions did not affect the viral growth on Marc-145 cells, but deletion of these regions led to earlier PRRSV replication increased (before 36 h after infectious in vitro). Conclusion The functional roles of nsp2 variable middle region for PRRSV HuN4-F112 replication have been identified. Our results also suggested that none-essential region might be an ideal insertion region to express foreign gene in PRRSV genome.