A candidate competitive ELISA based on monoclonal antibody 3A8 for diagnosis of contagious bovine pleuropneumonia.

For the development of a competitive ELISA (cELISA) to detect serum antibodies against the Mycoplasma mycoides subsp. Mycoides (Mmm) (strain PG1), the causative agent of contagious bovine pleuropneumonia (CBPP), all the proteins of this pathogen were analyzed. Then, a specific extracellular region of a transmembrane protein with the potential for diagnosis was identified. After that, a monoclonal antibody (Mab) named 3A8 was obtained using this extracellular region as an immunogen. Finally, a cELISA was established with the extracellular domain of this transmembrane protein as the coating antigen, Mab 3A8 as the competitive antibody, and HRP-labeled goat anti-mouse IgG as the enzyme-labeled antibody. This established method was used to detect the antibody dynamic regularity of goats which are artificially immunized Mmm and was also compared with a commercial ELISA kit. Further, the sera of 1011 different cattle from border provinces of China were monitored using a candidate Mab 3A8 cELISA. The detection results of known background sera used in this study indicate that a candidate diagnostic marker was successfully identified by analyzing all the coding proteins of Mmm in this research, and the cELISA established based on the Mab 3A8 against this protein can detect CBPP-positive serum with specificity and has no cross-reaction with other related epidemic disease-positive sera. In addition, we tested the sera collected from the border areas of China using the established ELISA, and no positive sample was detected. The research protocol of the CBPP cELISA established in this study is different from the traditional method, which can greatly reduce the investment of manpower and capital and save development time. We believe that this study's protocol could serve as a reference for the development of detection methods for mycoplasma and other complex pathogens. KEY POINTS: • A Mmm-specific diagnostic marker was obtained based on protein characteristics. • A cELISA was established for CBPP serum antibody detection. • The serological investigation was conducted for CBPP in the border areas of China.

Postoperative intermittent pneumatic compression for preventing venous thromboembolism in Chinese lung cancer patients: a randomized clinical trial.

BACKGROUND: Postoperative lung cancer patients belong to the high-risk group for venous thromboembolism (VTE). The standardized preventive measures for perioperative VTE in lung cancer are not perfect, especially for the prevention and treatment of catheter-related thrombosis (CRT) caused by carried central venous catheters (CVCs) in lung cancer surgery. PATIENTS AND METHODS: This study included 460 patients with lung cancer undergoing video-assisted thoracic surgery (VATS) in our center from July 2020 to June 2021. Patients were randomized into two groups, and intraoperatively-placed CVCs would be carried to discharge. During hospitalization, the control group was treated with low-molecular-weight heparin (LMWH), and the experimental group with LMWH + intermittent pneumatic compression (IPC). Vascular ultrasound was performed at three time points which included before surgery, before discharge, and one month after discharge. The incidence of VTE between the two groups was studied by the Log-binomial regression model. RESULTS: CRT occurred in 71.7% of the experimental group and 79.7% of the control group. The multivariate regression showed that the risk of developing CRT in the experimental group was lower than in the control group (Adjusted RR = 0.889 [95%CI0.799-0.989], p = 0.031), with no heterogeneity in subgroups (P for Interaction > 0.05). Moreover, the fibrinogen of patients in the experimental group was lower than control group at follow-up (P = 0.019). CONCLUSION: IPC reduced the incidence of CRT during hospitalization in lung cancer patients after surgery. TRIAL REGISTRATION: No. ChiCTR2000034511.

Development and validation of a mutation-based model to predict immunotherapeutic efficacy in NSCLC.

Background: Immunotherapy has become increasingly important in the perioperative period of non-small-cell lung cancer (NSCLC). In this study, we intended to develop a mutation-based model to predict the therapeutic effificacy of immune checkpoint inhibitors (ICIs) in patients with NSCLC. Methods: Random Forest (RF) classifiers were generated to identify tumor gene mutated features associated with immunotherapy outcomes. Then the best classifier with the highest accuracy served for the development of the predictive model. The correlations of some reported biomarkers with the model were analyzed, such as TMB, PD-(L)1, KEAP1-driven co-mutations, and immune subtypes. The training cohort and validation cohorts performed survival analyses to estimate the predictive efficiency independently. Results: An 18-gene set was selected using random forest (RF) classififiers. A predictive model was developed based on the number of mutant genes among the candidate genes, and patients were divided into the MT group (mutant gene ≥ 2) and WT group (mutant gene < 2). The MT group (N = 54) had better overall survival (OS) compared to the WT group (N = 290); the median OS was not reached vs. nine months (P < 0.0001, AUC = 0.73). The robust predictive performance was confifirmed in three validation cohorts, with an AUC of 0.70, 0.57, and 0.64 (P < 0.05). The MT group was characterized by high tumor neoantigen burden (TNB), increased immune infifiltration cells such as CD8 T and macrophage cells, and upregulated immune checkpoint molecules, suggesting potential biological advantages in ICIs therapy. Conclusions: The predictive model could precisely predict the immunotherapeutic efficacy in NSCLC based on the mutant genes within the model. Furthermore, some immune-related features and cell expression could support robust efficiency.

Immunogenicity and efficacy of serogroup A and D bacterins against Pasteurella multocida in mice.

Introduction: Pasteurella multocida is a widespread respiratory pathogen in pigs, causing swine pneumonia and atrophic rhinitis, and the capsular serogroups A and D are the main epidemic serogroups in infected animals. This study investigated the protective effects of serogroup A and D bacterins against current circulating P. multocida strains, to better understand the immunity generated by bacterins. Method: 13 serogroup A (seven A: L3 and six A: L6 strains) and 13 serogroup D (all D: L6 strains) P. multocida strains were isolated, and used as inactivated whole cell antigen to prepare P. multocida bacterins. Mice were immunized with these bacterins at 21-day interval and intraperitoneally challenged with the homologous and heterologous P. multocida strains, respectively. The antibody titer levels and immunization protective efficacy of vaccines were evaluated. Results: All of the bacterins tested induced high titer levels of immunoglobulin G antibodies against the parental bacterial antigen in mice. Vaccination with the six A: L6 bacterins provided no protection against the parent strain, but some strains did provide heterologous protection against A: L3 strains. Vaccination with the seven A: L3 bacterins provided 50%-100% protection against the parent strain, but none gave heterologous protection against the A:L6 strains. Immunization with the thirteen D: L6 bacterins offered 60%-100% protection against the parent strain, and almost all D: L6 strains gave cross-protection. Discussion: This study found that the cross-protectivity of serogroup A strains was poor, while serogroup D strains was effective, which provided some insights for P. multocida vaccine development.

Identification of three small nucleolar RNAs (snoRNAs) as potential prognostic markers in diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a non-Hodgkin lymphoma with high mortality rates. Small nucleolar RNAs (snoRNAs) are tumor-specific biological markers, but there are few studies on the role of snoRNAs in DLBCL. MATERIALS AND METHODS: Survival-related snoRNAs were selected to construct a specific snoRNA-based signature via computational analyses (Cox regression and independent prognostic analyses) to predict the prognosis of DLBCL patients. To assist in clinical applications, a nomogram was built by combining the risk model and other independent prognostic factors. Pathway analysis, gene ontology analysis, transcription factor enrichment, protein-protein interactions, and single nucleotide variant analysis were used to explore the potential biological mechanisms of co-expressed genes. RESULTS: Twelve prognosis-correlated snoRNAs were selected from the DLBCL patient cohort of microarray profiles, and a three-snoRNA signature consisting of SNORD1A, SNORA60, and SNORA66 was constructed. DLBCL patients could be divided into high-risk and low-risk cohorts using the risk model, and the high-risk group and activated B cell-like (ABC) type DLBCL were linked with disappointing survival. In addition, SNORD1A co-expressed genes were inseparably linked to the biological functions of the ribosome and mitochondria. Potential transcriptional regulatory networks have also been identified. MYC and RPL10A were the most mutated SNORD1A co-expressed genes in DLBCL. CONCLUSION: Put together, our findings explored the potential biological effects of snoRNAs in DLBCL, and provided a new predictor for DLBCL prediction.

Retracted: Britannin mediates apoptosis and glycolysis of T-cell lymphoblastic lymphoma cells by AMPK-dependent autophagy.

The above article, published online on 19 September 2022 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/abs/10.1002/jbt.23211), has been retracted by agreement between the authors, the journal Editor in Chief, Hari Bhat, and Wiley Periodicals, LLC. The article is being retracted at the authors' request because some of the data underlying this article refer to a different cell line from the one reported in it. As a result, the article's conclusions do not accurately reflect the full data and cannot be considered reliable.

Analysis on Damage and Mechanical Properties of Ballastless Track in a Tunnel after a Fire.

In order to explore the damage and mechanical properties of ballastless track after a fire, the uniaxial compressive strength, shear strength, peak strain, and elastic modulus changes due to temperature were obtained through uniaxial compressive and shear tests of concrete after exposure to high temperatures. The test results showed that with increases in temperature, the uniaxial compressive strength, shear strength, and elastic modulus of concrete all presented a decreasing trend, while the peak strain had an increasing trend. Then, based on the classical damage theory model and the strength probability distribution function of concrete micro-units, the high-temperature damage constitutive equation for concrete was established, and the compressive stress-strain curve of concrete after exposure to high temperature was reproduced. Finally, using the CFD numerical simulation software, the temperature field of a ballastless track structure in a tunnel during a fire was obtained, and the temperatures at different positions of ballastless track bed were acquired. Combined with the high-temperature damage constitutive equation for concrete deduced from tests and theoretical analysis, the strength and damage values of the ballastless track bed at different positions after a tunnel fire were obtained.

Sintilimab combined with chidamide in the treatment of extranodal nature killer/T-cell lymphoma with secondary hemophagocytic lymphohistiocytosis: Two case reports and literature review.

RATIONALE: Extranodal nature killer/T-cell lymphoma (ENKTL) failing in asparaginase-containing treatments is fatal, it has a higher mortality rate when accompanied by secondary hemophagocytic lymphohistiocytosis (HLH). The study reported 2 ENKTL-related HLH patients. PATIENT CONCERNS: Patient 1 visited for nasal congestion and runny nose for 6 months then got a fever and serious myelosuppression after P-GEP (pegaspargase, gemcitabine, etoposide, and methylprednisolone) chemotherapy. Patient 2 complained of painless lymphadenectasis in the right neck for 4 months and experienced recurrent fever and poor performance status after 3 cycles of P-Gemox (pegaspargase, gemcitabine, and oxaliplatin) chemotherapy. DIAGNOSES: Patient 1 and patient 2 were diagnosed as ENKTL failing in asparaginase-based chemotherapy and involving secondary HLH. INTERVENTIONS: The dose of chidamide was 20 mg twice a week for 2 weeks and sintilimab was 200 mg once every 3 weeks. OUTCOMES: ENKTL was relieved and the HLH was resolved after the therapy of sintilimab and chidamide. The patients had achieved durable survival without immune-related adverse events. LESSONS: ENKTL-related HLH needs early diagnosis and treatment. The combined strategy of sintilimab plus chidamide help deal with HLH and solve ENKTL, it may be a useful treatment option for ENKTL-related HLH.

Influence of temperature gradient of slab track on the dynamic responses of the train-CRTS III slab track on subgrade nonlinear coupled system.

Temperature is an important load for ballastless track. However, there is little research on the system dynamic responses when a train travels on a ballastless track under the temperature gradient of ballastless track. Considering the moving train, temperature gradient of slab track, gravity of slab track, and the contact nonlinearity between interfaces of slab track, a dynamic model for a high-speed train runs along the CRTS III slab track on subgrade is developed by a nonlinear coupled way in ANSYS. The system dynamic responses under the temperature gradient of slab track with different amplitudes are theoretically investigated with the model. The results show that: (1) The proportions of the initial force and stress caused by the temperature gradient of slab track are different for different calculation items. The initial fastener tension force and positive slab bending stress have large proportions exceeding 50%. (2) The maximum dynamic responses for slab track are not uniform along the track. The maximum slab bending stress, slab acceleration, concrete base acceleration appear in the slab middle, at the slab end, and at the concrete base end, respectively. (3) The maximum accelerations of track components appear when the fifth or sixth wheel passes the measuring point, and at least two cars should be used. (4) The temperature gradient of slab track has a small influence on the car body acceleration. However, the influences on the slab acceleration, concrete base acceleration, fastener tension force are large, and the influence on the slab bending stress is huge.

Mycoplasma hyopneumoniae membrane protein Mhp271 interacts with host UPR protein GRP78 to facilitate infection.

The unfolded protein response (UPR) plays a crucial role in Mycoplasma hyopneumoniae (M. hyopneumoniae) pathogenesis. We previously demonstrated that M. hyopneumoniae interferes with the host UPR to foster bacterial adhesion and infection. However, the underlying molecular mechanism of this UPR modulation is unclear. Here, we report that M. hyopneumoniae membrane protein Mhp271 interacts with host GRP78, a master regulator of UPR localized to the porcine tracheal epithelial cells (PTECs) surface. The interaction of Mhp271 with GRP78 reduces the porcine beta-defensin 2 (PBD-2) production, thereby facilitating M. hyopneumoniae adherence and infection. Furthermore, the R1-2 repeat region of Mhp271 is crucial for GRP78 binding and the regulation of PBD-2 expression. Intriguingly, a coimmunoprecipitation (Co-IP) assay and molecular docking prediction indicated that the ATP, rather than the substrate-binding domain of GRP78, is targeted by Mhp271 R1-2. Overall, our findings identify host GRP78 as a target for M. hyopneumoniae Mhp271 modulating the host UPR to facilitate M. hyopneumoniae adherence and infection.

Expression of N6-methyladenosine (m6A) regulators correlates with immune microenvironment characteristics and predicts prognosis in diffuse large cell lymphoma (DLBCL).

This study conducted a comprehensive analysis of the clinical significance of N6-methyladenosine (m6A) regulators and their relationship with immune microenvironment characteristics in diffuse large cell lymphoma (DLBCL). Consensus clustering was performed to molecularly discriminate DLBCL subtypesbased on m6A regulators' expression. Using the Cox and Lasso regression algorithm, survival-associated m6A regulators were identified, and a m6A-based prognostic signature was established. The influence of m6A risk on immune cell infiltration, immune checkpoint genes, cancer immunity cycle, and immunotherapeutic response was evaluated. Potential molecular pathways related to m6A risk were investigated using gene set enrichment analysis. The m6A regulators showed satisfactory performance in distinguishing DLBCL subgroups with distinct clinical traits and outcomes. A six m6A regulator-based prognostic signature was established and validated as an independent predictor, which separated patients into low- and high-risk groups. High-risk m6A indicated worse survival. The B cells naïve, T cells gamma delta, and NK cells resting were the three most affected immune cells by m6A risk. Up-regulated (PDCD1 and KIR3DL1) and down-regulated (TIGIT, IDO1, and BTLA) immune checkpoint genes in the high-risk group were identified. The m6A risk was found to influence several steps in the cancer immunity cycle. Patients with high-risk m6A were more likely to benefit from immunotherapy. Biological function enrichment analysis revealed that high-risk m6A to be tended related to malignant tumor characteristics, while low-risk m6A showed trend to be related to defensive response processes. Collectively, the m6A-based prognostic signature could be a practical prognostic predictor for DLBCL and immune microenvironment characteristics affected by m6A may be part of the mechanism.

Sequential P-GEMOX and radiotherapy for early-stage extranodal natural killer/T-cell lymphoma: A multicenter study.

Extranodal natural killer/T-cell lymphoma, nasal-type (ENKTL) is a distinct subtype of non-Hodgkin lymphoma and most of the patients presented localized disease. Combined modality therapy (CMT), namely chemotherapy combined with radiotherapy, has been recommended for patients with early-stage ENKTL. However, the optimal CMT has not been fully clarified. This study reports the efficacy and toxicity of sequential P-GEMOX (pegaspargase, gemcitabine and oxaliplatin) and radiotherapy in a large Chinese cohort comprising of 202 patients diagnosed with early-stage ENKTL from six medical centers. The observed best overall response rate was 96.0% and 168 (83.2%) patients achieved complete remission. With a median follow-up of 44.1 months, the 3-year progression-free survival (PFS) and overall survival (OS) were 74.6% and 85.2%, respectively. Multivariate analysis suggested that extensive primary tumor (PFS, hazard ratio [HR] 3.660, 95% CI 1.820-7.359, p < 0.001; OS, HR 3.825, 95% CI 1.442-10.148, p = 0.007) and Eastern Cooperative Oncology Group performance status ≥ 2 (PFS, 3.042, 95% CI 1.468-6.306, p = 0.003; OS, HR 3.983, 95% CI 1.678-9.457, p = 0.02) were independent prognostic factors for survival outcomes. Among the established prognostic models for ENKTL, the nomogram-revised risk index model had optimal prognostic risk stratification ability (PFS, p < 0.001; OS, p < 0.001) and relatively balanced population distribution. The adverse events of this CMT were well-tolerated and manageable. In conclusion, sequential P-GEMOX and radiotherapy showed favorable efficacy with acceptable toxicity, and could be an effective treatment option for early-stage ENKTL patients.

Influence of Vehicle Number on the Dynamic Characteristics of High-Speed Train-CRTS III Slab Track-Subgrade Coupled System.

In this paper, a high-speed train-CRTS III slab track-subgrade coupled dynamic model is established. With the model, the influence of vehicle number on the dynamic characteristics of a train-CRTS III slab track-subgrade coupled system with smooth and random track irregularity conditions for conventional and vibration-reduction CRTS III slab tracks are theoretically studied and analyzed. Some conclusions are drawn from the results: (1) the largest dynamic responses of the coupled system for all items and cases are no longer changed when the vehicle number exceeds three, and three vehicles are adequate to guarantee the simulation precision to investigate the dynamic responses of the coupled system. (2) The acceleration of the car body has almost no relation with the vehicle number, and only one vehicle is needed to study the vehicle dynamics using the train-CRTS III slab track-subgrade coupled dynamic model. (3) For the conventional CRTS III slab track on a subgrade, the vehicle number has a negligible influence on the accelerations of the rail, slab, and concrete base, the positive and negative bending moments of the rail, the compressive force of the fastener, and the positive bending stress of slab, but it has a large influence on the tension force of the fastener, and the negative bending stresses of the slab and concrete base. Only one vehicle is needed to study track dynamics without considering the tension force of the fastener, the negative bending stresses of the slab and concrete base, otherwise, two or more vehicles are required. (4) For vibration reduction of the CRTS III slab track on a subgrade, the number of vehicles has some influence on the dynamic responses of all track components, and at least two vehicles are required to investigate the track dynamics.

Recombinant bluetongue virus with hemagglutinin epitopes in VP2 has potential as a labeled vaccine.

Bluetongue (BT) is an arbovirus-borne disease of ruminants caused by bluetongue virus (BTV) that has the potential to have a serious economic impact. Currently available commercial vaccines include attenuated vaccines and inactivated vaccines, both of which have achieved great success in the prevention and control of BTV. However, these vaccines cannot distinguish between infected animals and immunized animals. To control outbreaks of BTV, the development of labeled vaccines is urgently needed. In this study, we used the plasmid-based reverse genetics system (RGS) of BTV to rescue four recombinant viruses in which HA (influenza hemagglutinin) tags were inserted at different sites of VP2. In vitro, the recombinant tagged viruses exhibited morphologies, plaque, and growth kinetics similar to the parental BTV-16, and expressed both VP2 and HA tag. Subsequently, the selected recombinant tagged viruses were prepared as inactivated vaccines to immunize IFNAR(-/-) mice and sheep, and serological detection results of anti-HA antibody provided discriminative detection. In summary, we used plasmid-based RGS to rescue BTV recombinant viruses with HA tags inserted into VP2, and detected several sites on VP2 that can accommodate HA tags. Some of the recombinant tagged viruses have potential to be developed into distinctive inactivated vaccines.

Development and optimization of a DNA-based reverse genetics systems for epizootic hemorrhagic disease virus.

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Reoviridae, and has a genome consisting of 10 linear double-stranded (ds) RNA segments. The current reverse genetics system (RGS) for engineering the EHDV genome relies on the use of in vitro-synthesized capped viral RNA transcripts. To obtain more-efficient and simpler RGSs for EHDV, we developed an entirely DNA (plasmid or PCR amplicon)-based RGS for viral rescue. This RGS enabled the rescue of infectious EHDV from BSR-T7 cells following co-transfection with seven helper viral protein expression plasmids and 10 cDNA rescue plasmids or PCR amplicons representing the EHDV genome. Furthermore, we optimized the DNA-based systems and confirmed that some of the helper expression plasmids were not essential for the recovery of infectious EHDV. Thus, DNA-based RGSs may offer a more efficient method of recombinant virus recovery and accelerate the study of the biological characteristics of EHDV and the development of novel vaccines.

HONN-Based Adaptive ILC for Pure-Feedback Nonaffine Discrete-Time Systems With Unknown Control Directions.

Nearly all adaptive control techniques require that the control directions of dynamical systems are known in advance. In this paper, for a class of pure-feedback nonaffine discrete-time systems with unknown control directions (UCDs), a high-order neural network (HONN)-based adaptive iterative learning control (ILC) approach is presented to address a repetitive tracking control issue. The implicit function theorem is adopted to cope with the difficulty resulting from the nonaffine structure of control input. Employing a discrete Nussbaum-type function in the neural network weight adaptation law to suit the UCD, an HONN is used to iteratively estimate the ideal control signals. In addition, a novel dead-zone method is developed in the HONN-based adaptive ILC algorithm to enhance its robustness against nonrepetitive desired trajectories and random uncertainties in iterative initial errors and external disturbance. Consequently, the system output, except at the initial n time instants, is demonstrated to asymptotically converge to an adjustable range of the desired trajectory along the iteration axis, while all of the system signals remain bounded during the entire ILC process. Two simulation examples show the feasibility of the adaptive ILC approach.

Isolation and Characterization of Fengycins Produced by Bacillus amyloliquefaciens JFL21 and Its Broad-Spectrum Antimicrobial Potential Against Multidrug-Resistant Foodborne Pathogens.

The continuing emergence and development of pathogenic microorganisms that are resistant to antibiotics constitute an increasing global concern, and the effort in new antimicrobials discovery will remain relevant until a lasting solution is found. A new bacterial strain, designated JFL21, was isolated from seafood and identified as B. amyloliquefaciens. The antimicrobial substance produced by B. amyloliquefaciens JFL21 showed low toxicity to most probiotics but exhibited strong antimicrobial activities against multidrug-resistant foodborne pathogens. The partially purified antimicrobial substance, Anti-JFL21, was characterized to be a multiple lipopeptides mixture comprising the families of surfactin, fengycin, and iturin. Compared with commercially available polymyxin B and Nisin, Anti-JFL21 not only could exhibit a wider and stronger antibacterial activity toward Gram-positive pathogens but also inhibit the growth of a majority of fungal pathogens. After further separation through gel filtration chromatography (GFC), the family of surfactin, fengycin, and iturin were obtained, respectively. The results of the antimicrobial test pointed out that only fengycin family presented marked antimicrobial properties against the indicators of L. monocytogenes, A. hydrophila, and C. gloeosporioides, which demonstrated that fengycins might play a major role in the antibacterial and antifungal activity of Anti-JFL21. Additionally, the current study also showed that the fengycins produced by B. amyloliquefaciens JFL21 not only maintained stable anti-Listeria activity over a broad pH and temperature range, but also remained active after treatment with ultraviolet sterilization, chemical reagents, and proteolytic enzymes. Therefore, the results of this study suggest the new strain and its antimicrobials are potentially useful in food preservation for the biological control of the multidrug-resistant foodborne pathogens.

PCR-based reverse genetics strategy for bluetongue virus recovery.

BACKGROUND: Bluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription. RESULT: In this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others. CONCLUSION: In this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.

Impaired cellular energy metabolism contributes to bluetongue-virus-induced autophagy.

Bluetongue virus (BTV) has been found to trigger autophagy to favor its replication, but the underlying mechanisms have not been clarified. Here, we show that cellular energy metabolism is involved in BTV-induced autophagy. Cellular ATP synthesis was impaired by BTV1 infection, causing metabolic stress, which was responsible for activation of autophagy, since the conversion of LC3 and aggregation of GFP-LC3 (autophagy markers) were suppressed when infection-caused energy depletion was reversed via MP (metabolic substrate) treatment. The reduced virus yields with MP further supported this view. Overall, our findings suggest that BTV1-induced disruption of cellular energy metabolism contributes to autophagy, and this provides new insights into BTV-host interactions.

Dissection and integration of the autophagy signaling network initiated by bluetongue virus infection: crucial candidates ERK1/2, Akt and AMPK.

Bluetongue virus (BTV), a complex double-stranded segmented RNA virus, has been found to initiate cellular autophagy for its own benefit. Here, with a view to understanding the underlying mechanisms, we first systematically dissected the exact signaling network in BTV-induced autophagy. We found that the activity of mTOR, a crucial pivot, was inhibited by BTV1 infection, subsequently leading to downstream p70S6K suppression and autophagy initiation. We then explored the upstream regulators of mTOR and analyzed their activities via a series of assays. We found BTV1-induced autophagy to be independent of the ERK1/2 signaling pathway. However, the BTV1-induced inhibition of PI3K/Akt was found to be partially responsible for mTOR inactivation and subsequent autophagy initiation. Furthermore, we found unexpectedly that AMPK seemed to play a more important role in BTV1-induced autophagy. Elevated [Ca(2+)]cyto-mediated activation of CaMKKß exactly managed the activation of AMPK, which then positively regulated autophagy through suppressing mTOR. We must emphasize that TSC2 is a fatal mediator between upstream Akt or AMPK and downstream mTOR through its phosphorylation. Taken together, our data suggested that the BTV1-induced inhibition of the Akt-TSC2-mTOR pathway and the upregulation of the AMPK-TSC2-mTOR pathway both contributed to autophagy initiation and further favored virus replication.