Complete genome sequence of a novel highly pathogenic porcine reproductive and respiratory syndrome virus variant.

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) emerged in China in 2006, and HP-PRRS virus (HP-PRRSV) has evolved continuously. Here, the complete genomic sequence of a novel HP-PRRSV field strain, JX, is reported. The present finding will contribute to further studies focusing on the evolutionary mechanism of PRRSV.

Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-κB signaling pathway.

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects macrophages/dendritic cells and modulates cytokine expression in these cells. Interleukin-15 (IL-15) is a pleiotropic cytokine involved in wide range of biological activities. It has been shown to be essential for the generation, activation, and proliferation of NK and NKT cells and for the survival and activation of CD8(+) effector and memory T cells. In this study, we discovered that PRRSV infection upregulated IL-15 production at both the mRNA and protein levels in porcine alveolar macrophages (PAMs), blood monocyte-derived macrophages (BMo), and monocyte-derived dendritic cells (DCs). We subsequently demonstrated that the NF-κB signaling pathway was essential for PRRSV infection-induced IL-15 production. First, addition of an NF-κB inhibitor drastically reduced PRRSV infection-induced IL-15 production. We then found that NF-κB was indeed activated upon PRRSV infection, as evidenced by IκB phosphorylation and degradation. Moreover, we revealed an NF-κB binding motif in the cloned porcine IL-15 (pIL-15) promoter, deletion of which abrogated the pIL-15 promoter activity in PRRSV-infected alveolar macrophages. In addition, we demonstrated that PRRSV nucleocapsid (N) protein had the ability to induce IL-15 production in porcine alveolar macrophage cell line CRL2843 by transient transfection, which was mediated by its multiple motifs, and it also activated NF-κB. These data indicated that PRRSV infection-induced IL-15 production was likely through PRRSV N protein-mediated NF-κB activation. Our findings provide new insights into the molecular mechanisms underling the IL-15 production induced by PRRSV infection.

MCL-1 Inhibitor S63845 Distinctively Affects Intramedullary and Extramedullary Hematopoiesis.

Conventional chemotherapy for killing cancer cells using cytotoxic drugs suffers from low selectivity, significant toxicity, and a narrow therapeutic index. Hyper-specific targeted drugs achieve precise destruction of tumors by inhibiting molecular pathways that are critical to tumor growth. Myeloid cell leukemia 1 (MCL-1), an important pro-survival protein in the BCL-2 family, is a promising antitumor target. In this study, we chose to investigate the effects of S63845, a small-molecule inhibitor that targets MCL-1, on the normal hematopoietic system. A mouse model of hematopoietic injury was constructed, and the effects of the inhibitor on the hematopoietic system of mice were evaluated via routine blood tests and flow cytometry. The results showed that S63845 affected the hematopoiesis of various lineages in the early stage of action, causing extramedullary compensatory hematopoiesis in the myeloid and megakaryocytic lineages. The maturation of the erythroid lineage in the intramedullary and extramedullary segments was blocked to varying degrees, and both the intramedullary and extramedullary lymphoid lineages were inhibited. This study provides a complete description of the effects of MCL-1 inhibitor on the intramedullary and extramedullary hematopoietic lineages, which is important for the selection of combinations of antitumor drugs and the prevention of adverse hematopoiesis-related effects.

Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche.

Limited numbers of available hematopoietic stem cells (HSCs) limit the widespread use of HSC-based therapies. Expansion systems for functional heterogenous HSCs remain to be optimized. Here, we present a convenient strategy for human HSC expansion based on a biomimetic Microniche. After demonstrating the expansion of HSC from different sources, we find that our Microniche-based system expands the therapeutically attractive megakaryocyte-biased HSC. We demonstrate scalable HSC expansion by applying this strategy in a stirred bioreactor. Moreover, we identify that the functional human megakaryocyte-biased HSCs are enriched in the CD34+CD38-CD45RA-CD90+CD49f lowCD62L-CD133+ subpopulation. Specifically, the expansion of megakaryocyte-biased HSCs is supported by a biomimetic niche-like microenvironment, which generates a suitable cytokine milieu and supplies the appropriate physical scaffolding. Thus, beyond clarifying the existence and immuno-phenotype of human megakaryocyte-biased HSC, our study demonstrates a flexible human HSC expansion strategy that could help realize the strong clinical promise of HSC-based therapies.

Induction of interleukin-10 is dependent on p38 mitogen-activated protein kinase pathway in macrophages infected with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory illness in pigs and usually establishes a persistent infection. Previous studies suggested that interleukin-10 (IL-10) could play a critical role in PRRSV-induced immunosuppression. However, the ability of PRRSV to induce IL-10 in infected cells is controversial. In this study, we further investigated this issue using PRRSV strain CH-1a, which is the first North American genotype strain isolated in China. RESULTS: PRRSV strain CH-1a could significantly up-regulate IL-10 production both at mRNA and protein levels in porcine alveolar macrophages (PAMs), bone marrow-derived macrophages (BMDMs), and monocyte-derived macrophages (MDMs). However, up-regulation of IL-10 by PRRSV was retarded by specific inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580) and NF-κB (BAY11-7082). Additionally, p38 MAPK and NF-κB pathways but not ERK1/2 MAPK were actually activated in PRRSV-infected BMDMs as demonstrated by western blot analysis, suggesting that p38 MAPK and NF-κB pathways are involved in the induction of IL-10 by PRRSV infection. Transfection of PAMs and PAM cell line 3D4/21 (CRL-2843) with viral structural genes showed that glycoprotein5 (GP5) could significantly up-regulate IL-10 production, which was dependent on p38 MAPK and signal transducer and activator of transcription-3 (STAT3) activation. We also demonstrated that a full-length glycoprotein was essential for GP5 to induce IL-10 production. CONCLUSIONS: PRRSV strain CH-1a could significantly up-regulate IL-10 production through p38 MAPK activation.

Levistilide A Promotes Expansion of Human Umbilical Cord Blood Hematopoietic Stem Cells by Enhancing Antioxidant Activity.

Several approaches to expand human hematopoietic stem cells (hHSCs) clinically along with retainable capability of multipotential differentiation have been reported, but only a few have advanced to evaluation in clinical trials, which limits the application of HSC-based therapy. Here we show a phthalide derivative, Levistilide A (LA), can serve as a promising molecule to expand functional human umbilical cord blood (UCB) HSCs ex vivo. An in-house screen identified LA out of nine natural products as an outstanding candidate for hHSCs expansion. Additionally, our data indicated that LA treatment not only increased the numbers of phenotype-defined HSCs, but also enhanced their colony formation ability. Xenotransplantation assays showed that LA treatment could maintain unaffected engraftment of hHSCs with multilineage differentiation capacity. Further experiments revealed that LA enhanced the antioxidant activity of hHSCs by reducing intracellular and mitochondrial reactive oxygen species (ROS) levels. The identification of LA provides a new strategy in solving the clinical issue of limited numbers of UCB HSCs.

KDM4 inhibitor SD49-7 attenuates leukemia stem cell via KDM4A/MDM2/p21CIP1 axis.

Rationale: Traditional treatments for leukemia fail to address stem cell drug resistance characterized by epigenetic mediators such as histone lysine-specific demethylase 4 (KDM4). The KDM4 family, which acts as epigenetic regulators inducing histone demethylation during the development and progression of leukemia, lacks specific molecular inhibitors. Methods: The KDM4 inhibitor, SD49-7, was synthesized and purified based on acyl hydrazone Schiff base. The interaction between SD49-7 and KDM4s was monitored in vitro by surface plasma resonance (SPR). In vitro and in vivo biological function experiments were performed to analyze apoptosis, colony-formation, proliferation, differentiation, and cell cycle in cell sub-lines and mice. Molecular mechanisms were demonstrated by RNA-seq, ChIP-seq, RT-qPCR and Western blotting. Results: We found significantly high KDM4A expression levels in several human leukemia subtypes. The knockdown of KDM4s inhibited leukemogenesis in the MLL-AF9 leukemia mouse model but did not affect the survival of normal human hematopoietic cells. We identified SD49-7 as a selective KDM4 inhibitor that impaired the progression of leukemia stem cells (LSCs) in vitro. SD49-7 suppressed leukemia development in the mouse model and patient-derived xenograft model of leukemia. Depletion of KDM4s activated the apoptosis signaling pathway by suppressing MDM2 expression via modulating H3K9me3 levels on the MDM2 promoter region. Conclusion: Our study demonstrates a unique KDM4 inhibitor for LSCs to overcome the resistance to traditional treatment and offers KDM4 inhibition as a promising strategy for resistant leukemia therapy.

An aptamer-based effective method for highly sensitive detection of chloramphenicol residues in animal-sourced food using real-time fluorescent quantitative PCR.

Chloramphenicol (CAP) residues can not only harm human health through entering food chain, but also cause the spreading of drug-resistant bacteria, thereby leading to secondary environmental pollution. Therefore, it is in urgent need of establishing an efficient technology to detect CAP residues in animal-sourced food. In this study, a novel sensitive approach for detection of CAP was designed based on a CAP specific aptamer and real-time fluorescent quantitative PCR (qRT-PCR). The CAP specific aptamer was firstly hybridized with a biotin modified complementary probe, and then was immobilized on streptavidin conjugated magnetic beads through biotin. When CAP was added, the aptamer would specifically bind with CAP by forming a hairpin structure and be released from the magnetic beads for CAP detection by qRT-PCR. Factors (i.e., probe strand length, aptamer concentration, NaCl concentration and incubation time) that would influence the determination accuracy of this aptamer-based detection system were optimized. Under the optimized conditions, the present detection system exhibited a high sensitivity toward CAP with a limit of detection of 0.1ng/mL (linear range from 0.1 to 20ng/mL). Moreover, this detection system also showed high selectivity against thiamphenicol (TAP) and florfenicol (FF), which are CAP's structure analogs. Eventually, this detection system was applied for detecting CAP in real spiked milk. The recovery rate of CAP from spiked milk samples ranged from 94.0-102.0%. These results indicated this developed detection system a promising high sensitive and specific method of CAP residues detection in animal-sourced food.

In vitro effects of recombinant zebrafish IFN on spring viremia of carp virus and infectious hematopoietic necrosis virus.

In order to evaluate the effects of fish recombinant interferon (rIFN) on fish pathogenic rhabdoviruses, the zebrafish (Danio rerio) IFN (DreIFN) allele B gene was cloned and expressed in Escherichia coli. In addition, the effects of recombinant DreIFN (rDreIFN) on spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV), and vesicular stomatitis virus (VSV) were surveyed in fish and chicken cells. The mature peptide of DreIFN allele B gene encodes 163 amino acids. Residues 3 and 98 are a pair of cysteines that likely form an intrachain disulfide bridge. rDreIFN protein was detected as a band at 21.6 kDa by SDS-PAGE. The purified rDreIFN has anti-SVCV and anti-IHNV activity of 3 x 10(4) U/mg-10(7) U/mg. The results indicate that rDreIFN has higher activity against SVCV and IHNV on epithelioma papulosum cyprinid (EPC) than on grass carp (C. idellus) ovary (CO) cell lines and no activity against VSV on chick embryo fibroblasts (CEF).

Selection and Identification of Chloramphenicol-Specific DNA Aptamers by Mag-SELEX.

Chloramphenicol (CAP) has been widely used to treat bacterial infections in livestock and aquatic animals. To reduce the risk of CAP residues, an efficient technology to rapidly detect CAP residues in animal-sourced food is expressly needed. In this study, magnetic bead-based systematic evolution of ligands by exponential enrichment (Mag-SELEX) strategy was performed to select and identify CAP-specific single-stranded DNA (ssDNA) aptamers from a random oligonucleotide library. After nine rounds of selection, five potential ssDNA aptamers were selected. Low homology indicated that they might belong to different families. To identify an aptamer with the highest affinity for CAP, the dissociation constant (K d) values of these selected aptamers were determined. The lowest K d values of two potential aptamers (i.e., No. 4 and No. 5) were, respectively, 0.10162 ± 0.0111 and 0.03224 ± 0.00819 µM, which were much lower than previously reported lowest K d value (i.e., 0.766 µM) of CAP aptamer. Moreover, compared with No. 4, aptamer No. 5 had higher binding rate, which is quite different among those with CAP and with CAP's structural analogs (i.e., thiamphenicol (TAP) and florfenicol (FF)). These results indicated that the potential aptamer No. 5 with highest specificity and affinity for CAP would be an ideal aptamer for future detection of residual CAP in animal-sourced food.

Attenuation of highly pathogenic porcine reproductive and respiratory syndrome virus by inserting an additional transcription unit.

Transcription regulatory sequences (TRSs) play a key role in the synthesis of porcine reproductive and respiratory syndrome virus (PRRSV) subgenomic mRNAs, which resembles similarity-assisted RNA recombination. In this study, genome instability was found when a highly pathogenic PRRSV (HP-PRRSV) strain was inserted by an additional transcription unit in which a foreign gene GFP was expressed from TRS2 while a copy of TRS6 drove ORF2a/b transcription. Structural protein gene-deleted genomes resulted from enhanced RNA recombinations were identified in the recombinant virus rHV-GFP. Moreover, rHV-GFP replicated slower than parental viruses, and caused less cell death in porcine alveolar macrophages. Pigs infected with rHV-GFP survived with no or mild syndromes, whereas all pigs infected with parental viruses died within 12 days. Our data showed that additional transcription unit insertion could confer genome instability and attenuation of HP-PRRSV.

Highly pathogenic porcine reproductive and respiratory syndrome virus impairs LPS- and poly(I:C)-stimulated tumor necrosis factor-alpha release by inhibiting ERK signaling pathway.

Atypical porcine reproductive and respiratory syndrome (PRRS) characterized by high morbidity and mortality emerged in China in 2006. The causative agent was confirmed to be a highly pathogenic PRRS virus (HP-PRRSV). However, the pathogenesis of HP-PRRSV is still uncertain. Here, the ability of the highly pathogenic strains (HV and JX) to induce tumor necrosis factor alpha (TNF-α) was studied. Our results showed that HV and JX were weaker inducers of TNF-α than the conventional strain CH-1a. Moreover, HV infection was demonstrated to suppress extracellular signal-regulated kinase (ERK) phosphorylation at the early time points. Pharmacologic inhibition or activation of ERK revealed that TNF-α production in HV-infected macrophages was associated with the activation status of ERK. Furthermore, HV- and JX-infection could potently impair lipopolysaccharide (LPS)- and poly(I:C)-stimulated TNF-α release in a dose dependent manner whereas synergistic effects were observed at mRNA level. The observation suggested the involvement of posttranslational impact of HP-PRRSV on TNF-α production, which might be attributed to the reduced ERK1/2 phosphorylation in response to toll-like receptor (TLR)-ligation. Taken together, our results indicated that HP-PRRSV infection could impair TNF-α production by inhibiting ERK signaling pathway, which might partially contribute to the pathogenesis of HP-PRRSV.

Increase of CD163 but not sialoadhesin on cultured peripheral blood monocytes is coordinated with enhanced susceptibility to porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism mainly for porcine alveolar macrophages (PAMs), but not for peripheral blood monocytes (BMo) in vivo. Previous research showed that only a few BMo became susceptible to PRRSV infection after 1 day culture. Porcine sialoadhesin (PoSn) and CD163 are identified to be the two main PRRSV receptors for binding and internalization. Both receptors are not expressed on BMo, or only expressed at low levels, which may explain why PRRSV cannot infect them. The relationship of BMo differentiation/aging, PRRSV receptor level, and susceptibility to PRRS virus infection has not been thoroughly investigated. In this study, BMo were successfully cultured with pig serum plus L929 cell culture supernatant. Our results showed that both the mRNA and protein expression levels of PoSn were significantly increased after 5-day culture. The mRNA level of CD163 was enhanced more than 20-fold after 1-day culture; CD163-positive BMo increased dramatically from about 2% after 2h- culture to about 50% after 96-h culture. Furthermore, cultured BMo became much more permissive to PRRSV infection, and the percentage of PRRSV-infected BMo was at least the same as PAMs, if not higher, when infected with CH-1a, the first PRRSV strain isolated in China, or HV, a highly virulent strain. Three other PRRSV strains including VR2332, and two classical Chinese isolates could also infect cultured BMo as well. Most importantly, PRRS virus was successfully isolated from 14 of 15 antibody-positive serum samples using cultured BMo. These results suggest that the enhanced susceptibility of cultured BMo to PRRS virus is coordinated with increased CD163 expression, but less related to the delayed (day 5) increased expression of PoSn. Thus, cultured BMo could be an alternative choice for PRRS virus isolation and identification.

[Development and application of real-time quantitative RT- PCR assay for the detection of hepatitis E virus].

Hepatitis E virus (HEV) sequences including four major genotypes representative strains available in GenBank were aligned with the DNAMAN software. The highly conserved internal region of ORF2 was then subjected to design primers and a probe. Furthermore, a 0.3 kb fragment of HEV ORF2 containing the amplification region was transcribed in vitro to synthese cRNA standard and a universal real-time TaqMan PCR assay was optimized and developed to detect and quantify main genotypes RNA of HEV. The specificity and reliability of the real-time RT-PCR was confirmed by testing genotype I HEV, genotype IV HEV and clinical samples. The detection limit of real-time RT-PCR was found 2.0 x 10(1) copies per reaction using in vitro transcribed cRNA. Compared with nested RT-PCR in diagnosis of HEV, the real-time RT-PCR developed was 10 to 100-fold more sensitive than the nested RT-PCR. The detection results from 54 clinical specimens indicated real-time RT-PCR was a rapid, sensitive and reproducible diagnostic method for HEV. This assay will be useful as an early and rapid diagnostic assay for HEV.

Thymocyte and peripheral blood T lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus.

Piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) are born severely immunocompromised. In this article we more closely examine the effects of in utero PRRSV infection on circulating and thymic T cell populations. Numbers of CD4+, CD8+, and dual-positive lymphocytes were quantitated in circulation and in the thymus during the 2 weeks following birth. At birth we found that the number of circulating lymphocytes was suppressed by 60%. Lymphocyte numbers were also suppressed by 42% at day 7, but by day 14 the number of lymphocytes had rebounded and was actually 47% greater than controls. At birth and day 7, a drop in the number of CD4+ cells could partially explain the suppression we observed, while the rebound in total lymphocyte numbers seen at day 14 was due to a nearly fourfold increase in the number of circulating CD8+ cells. As a result, the normal CD4+:CD8+ ratio of between 1.4 and 2.2 for neonatal pigs was reduced to 0.1-0.5. The thymuses of infected piglets were found to be 50% smaller than those of control pigs and were characterized by cortical involution and severe cortical depletion of thymocytes. Analysis of the population of thymocytes revealed that double-positive thymocytes were suppressed to a greater degree than either single positive subpopulation. In addition, we show that the number of thymocytes undergoing apoptosis was increased twofold in piglets infected with PRRSV. Taken together, these results help explain the dramatic immunosuppression observed in neonatal animals infected in utero with PRRSV.