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Mycoplasmas are minimal but notorious bacteria that infect humans and animals. These genome-reduced organisms have evolved strategies to overcome host apoptotic defense and establish persistent infection. Here, using Mycoplasma bovis as a model, we demonstrate that mycoplasma glycine cleavage system (GCS) H protein (GcvH) targets the endoplasmic reticulum (ER) to hijack host apoptosis facilitating bacterial infection. Mechanically, GcvH interacts with the ER-resident kinase Brsk2 and stabilizes it by blocking its autophagic degradation. Brsk2 subsequently disturbs unfolded protein response (UPR) signaling, thereby inhibiting the key apoptotic molecule CHOP expression and ER-mediated intrinsic apoptotic pathway. CHOP mediates a cross-talk between ER- and mitochondria-mediated intrinsic apoptosis. The GcvH N-terminal amino acid 31-35 region is necessary for GcvH interaction with Brsk2, as well as for GcvH to exert anti-apoptotic and potentially pro-infective functions. Notably, targeting Brsk2 to dampen apoptosis may be a conserved strategy for GCS-containing mycoplasmas. Our study reveals a novel role for the conserved metabolic route protein GcvH in Mycoplasma species. It also sheds light on how genome-reduced bacteria exploit a limited number of genomic proteins to resist host cell apoptosis thereby facilitating pathogenesis.
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Apoptosis , Proteínas Bacterianas , Retículo Endoplásmico , Humanos , Retículo Endoplásmico/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Animales , Infecciones por Mycoplasma/metabolismo , Infecciones por Mycoplasma/microbiología , Mycoplasma bovis/metabolismo , Glicina/metabolismo , Respuesta de Proteína Desplegada , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
This study aims to investigate the clinical characteristics of infections following induction chemotherapy for acute myeloid leukemia (AML) in children and identify risk factors associated with severe infections. Newly diagnosed children with AML treated at the Hematology Oncology Center of Beijing Children's Hospital affiliated to Capital Medical University (referred to as the "Beijing ward") and Baoding Hospital of Beijing Children's Hospital (referred to as the "Baoding ward") between November 2019 and August 2022 were enrolled. The diagnosis and treatment of the patients were carried out in accordance with the CCLG-AML 2019 protocol. Their essential information and infection-related indicators were collected. The various indicators between the two centers were compared. The incidence of infection in children with AML was 100%, with a severe infection rate of 15.3% and an infection-related mortality rate of 2.4%. Respiratory infections accounted for 39.7% of all infections events, and unspecified site infection for 32.2%. Bacterial infections were predominant at 51.2%. The bed unit area (BUA) varied significantly with 4.1 m2 in the Beijing ward and 10 m2 in the Baoding ward. Significant differences were observed in gastrointestinal infections (P < 0.001) and severe infections (P = 0.014) between the two wards. Several factors were identified as risk factors for severe infections, including BUA difference (OR = 4.353, 95% CI: 1.078-17.578), time of entering neutropenia phase after chemotherapy (OR = 6.369, 95% CI: 1.713-23.675) and bloodstream infection (OR = 7.466, 95% CI: 1.889-29.507). Respiratory tract infections and infections of unspecified site are most common during induction phase for pediatric AML. Bacteria, especially G- bacteria, are the leading pathogens. Risk factors for severe infections include a small BUA, entering neutropenia phase ≤ 5.5 days after chemotherapy, and bloodstream infection. Recognizing these risk factors can aid in the early identification and intervention of severe infections.
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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.
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Anticuerpos Monoclonales , Pleuroneumonía , Animales , Bovinos , Proteínas de la Membrana , China , Ensayo de Inmunoadsorción Enzimática , CabrasRESUMEN
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.
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Mycoplasma hyopneumoniae , Adhesinas Bacterianas/metabolismo , Animales , Chaperón BiP del Retículo Endoplásmico , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Simulación del Acoplamiento Molecular , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/metabolismo , Porcinos , Respuesta de Proteína DesplegadaRESUMEN
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.
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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.
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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.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Asparaginasa/uso terapéutico , Desoxicitidina/análogos & derivados , Linfoma Extranodal de Células NK-T/tratamiento farmacológico , Linfoma Extranodal de Células NK-T/radioterapia , Polietilenglicoles/uso terapéutico , Adulto , Anciano , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Asparaginasa/efectos adversos , Desoxicitidina/efectos adversos , Desoxicitidina/uso terapéutico , Femenino , Humanos , Linfoma Extranodal de Células NK-T/diagnóstico , Masculino , Persona de Mediana Edad , Compuestos Organoplatinos/efectos adversos , Compuestos Organoplatinos/uso terapéutico , Polietilenglicoles/efectos adversos , Pronóstico , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
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.
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Virus de la Enfermedad Hemorrágica Epizoótica/genética , Genética Inversa/métodos , Virología/métodos , Animales , Línea Celular , ADN Complementario/genética , Virus de la Enfermedad Hemorrágica Epizoótica/crecimiento & desarrollo , Mesocricetus , Plásmidos , ARN Viral/genética , Recombinación Genética , Infecciones por Reoviridae/virologíaRESUMEN
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.
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Virus de la Lengua Azul/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Genética Inversa/métodos , Virología/métodos , Animales , Virus de la Lengua Azul/genética , Línea Celular , Cricetinae , Viabilidad Microbiana , Plásmidos , TransfecciónRESUMEN
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.
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Autofagia , Virus de la Lengua Azul/fisiología , Metabolismo Energético , Replicación Viral , Animales , Virus de la Lengua Azul/patogenicidad , Células Cultivadas , CricetinaeRESUMEN
Epizootic haemorrhagic disease is a non-contagious infectious viral disease of wild and domestic ruminants caused by epizootic hemorrhagic disease virus (EHDV). EHDV belongs to the genus Orbivirus within the family Reoviridae and is transmitted by insects of the genus Culicoides. The impact of epizootic haemorrhagic disease is underscored by its designation as a notifiable disease by the Office International des Epizooties. The EHDV genome consists of 10 linear dsRNA segments (Seg1-Seg10). Until now, no reverse genetics system (RGS) has been developed to generate replication-competent EHDV entirely from cloned cDNA, hampering detailed functional analyses of EHDV biology. Here, we report the generation of viable EHDV entirely from cloned cDNAs. A replication-competent EHDV-2 (Ibaraki BK13 strain) virus incorporating a marker mutation was rescued by transfection of BHK-21 cells with expression plasmids and in vitro synthesized RNA transcripts. Using this RGS, two additional modified EHDV-2 viruses were also generated: one that contained a duplex concatemeric Seg9 gene and another that contained a duplex concatemeric Seg10 gene. The modified EHDV-2 with a duplex Seg9 gene was genetically stable during serial passage in BHK-21 cells. In contrast, the modified EHDV-2 with a duplex Seg10 gene was unstable during serial passage, but displayed enhanced replication kinetics in vitro when compared with the WT virus. This RGS provides a new platform for the investigation of EHDV replication, pathogenesis and novel EHDV vaccines.
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Enfermedades de los Bovinos/virología , Duplicación de Gen , Reordenamiento Génico , Virus de la Enfermedad Hemorrágica Epizoótica/genética , Infecciones por Reoviridae/veterinaria , Genética Inversa/métodos , Animales , Bovinos , Virus de la Enfermedad Hemorrágica Epizoótica/aislamiento & purificación , Virus de la Enfermedad Hemorrágica Epizoótica/fisiología , Filogenia , Infecciones por Reoviridae/virología , Replicación ViralRESUMEN
Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. We have previously reported that BTV1 infection induced autophagy for its own benefit, but how this occurs remains unclear. Here, the classical autophagy features including autophagsomes formation, GFP-LC3 dots and LC3-II conversation were shown in BTV1-infected cells, we also found the endoplasmic reticulum (ER) stress was triggered by BTV1 infection, which was demonstrated by the increased transcription level of the ER stress marker GRP78 and the expanded morphology of ER. During ER stress, PERK and eIF2α phosphorylation increased along with BTV1 infection, consistent with the elevated LC3 level, indicating that the PERK pathway of the unfolded protein response (UPR) was activated. In addition, both the blockage of PERK by GSK2656157 or knockdown of eIF2α by siRNA reduced the level of LC3, which suggested that the PERK-eIF2α pathway contributed to autophagy induced by BTV1. Furthermore, inactivation of PERK or silencing of eIF2α both significantly reduced the expression of VP2 protein and the viral yields in the supernatants. In sum, these data suggest that ER stress mediates autophagy via the PERK-eIF2α pathway and contributes to BTV1 replication, thus offering new insight into the molecular mechanisms of the BTV-host interaction.
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Virus de la Lengua Azul/patogenicidad , Factor 2 Eucariótico de Iniciación/metabolismo , eIF-2 Quinasa/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagia , Virus de la Lengua Azul/genética , Virus de la Lengua Azul/fisiología , Proteínas de la Cápside/genética , Proteínas de la Cápside/fisiología , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2 Eucariótico de Iniciación/genética , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/metabolismo , Indoles/farmacología , Modelos Biológicos , Transducción de Señal , Respuesta de Proteína Desplegada , Replicación Viral , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
BACKGROUND: The non-structural protein 3 (NS3) of bluetongue virus (BTV) is the second smaller non-structural protein produced in host cells, playing an important role in BTV trafficking and release. RESULTS: In this study, we generated five BTV NS3-reactive monoclonal antibodies (mAbs), named 3D8, 2G9, 1B5, 4H8, and 2B12. A panel of overlapping NS3-derived peptides representing the entirety of the BTV15 NS3 protein was screened to identify linear peptide epitopes recognized by each mAb. Based on the initial screen, a series of progressively truncated peptides were produced to identify the minimal linear peptide sequence required to maintain mAb binding. We found that mAb 3D8 reacted with the motif (36)PPRYA(40), 2G9 reacted with the motif (82)AEAFRDDVRLRQIK(95), 1B5 reacted with the motif (205)YNDAVRMSF(213), 2B12 and 4H8 reacted with the motif (204)SYNDAVRMSF(213). Sequence alignments demonstrated that these linear epitopes are highly conserved among all BTV serotypes, consistent with the observation that each mAb was able to recognize cells infected with BTV1-24 serotypes tested and each identified B cell epitope was able to be recognized by BTV-infect sheep serum. CONCLUSION: This collection of mAbs along with defined linear epitopes may provide useful reagents for investigations of NS3 protein function and the development of BTV group-specific diagnostics.
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Virus de la Lengua Azul/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Proteínas no Estructurales Virales/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Secuencia Conservada , Análisis Mutacional de ADNRESUMEN
Bluetongue virus (BTV) is the etiological agent of bluetongue (BT) disease, a noncontagious insect-transmitted disease of international importance. To date, 26 BTV serotypes have been recognized worldwide. Methods to discriminate BTV serotypes in clinical samples are essential to epidemiological surveillance efforts and BTV vaccination programs. The BTV VP2 major outer capsid protein, encoded by genomic segment 2 (Seg-2), is the most highly variable BTV protein and is the primary determinant of the virus serotype. Here, we report the development of rapid and reliable real-time RT-PCR assays to detect and discriminate 22 BTV serotypes on the basis of VP2-encoding genomic sequences. Serotype-specific primers and probes detected only the targeted BTV serotype and displayed no cross-amplification of off-target BTV serotypes or other closely related Reoviridae and Bunyaviridae family members. The real-time RT-PCR assays developed were highly sensitive, and the majority of serotype-specific reactions could detect template when present at ≥10 copies. These BTV serotype-specific real-time RT-PCR assays represent a rapid, sensitive, and reliable method for the identification, differentiation and quantification of 22 BTV serotypes.
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Virus de la Lengua Azul/clasificación , Lengua Azul/virología , Genotipo , Técnicas de Genotipaje/métodos , Sondas de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Virus de la Lengua Azul/genética , Sensibilidad y Especificidad , Serogrupo , OvinosRESUMEN
Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.
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Virus de la Lengua Azul/inmunología , Lengua Azul/prevención & control , Esquemas de Inmunización , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Lengua Azul/inmunología , Virus de la Lengua Azul/genética , Portadores de Fármacos , Virus de la Viruela de las Aves de Corral/genética , Ratones , Ovinos , Resultado del Tratamiento , Vacunas de ADN/genética , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/genéticaRESUMEN
Many approaches for generating large quantities of recombinant protein in Escherichia coli fuse the protein of interest to a protein tag to enhance solubility and improve recovery. However, the fusion tags can confound downstream applications, as the fusion partner can alter the structure and biological activity of the recombinant protein and proteolytic removal of the fusion tags can be expensive. Here we describe a new system for production of native proteins in E. coli that allows for removal of the fusion tag via intracellular self-cleavage by the human rhinovirus 3C (HRV3C) protease. This system allows for parallel cloning of target protein coding sequences into six different expression vectors, each with a different fusion partner tag to enhance solubility during induction. Temperature-regulated expression of the HRV3C protease allows for intracellular removal of the fusion tag following induction, and the liberated recombinant protein can be purified by affinity chromatography by virtue of a short six-histidine tag. This system will be an attractive approach for the expression and purification of recombinant proteins free of solubility-enhancing fusion tags, and should be amenable to high-throughput applications.
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Clonación Molecular/métodos , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteasas Virales 3C , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Escherichia coli/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Recombinantes de Fusión/genética , Solubilidad , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Smallpox is a highly contagious human disease caused by the variola virus. Although the disease was eliminated in 1979 due to its highly contagious nature and historical pathogenicity, with a mortality rate of up to 30%, this virus is an important candidate for biological weapons. Currently, vaccines are the critical measures to prevent this virus infection and spread. In this study, we designed a peptide vaccine using immunoinformatics tools, which have the potential to activate human immunity against variola virus infection efficiently. The design of peptides derives from vaccine-candidate proteins showing protective potential in vaccinia WR strains. Potential non-toxic and nonallergenic T-cell and B-cell binding and cytokine-inducing epitopes were then screened through a priority prediction using special linkers to connect B-cell epitopes and T-cell epitopes, and an appropriate adjuvant was added to the vaccine construction to enhance the immunogenicity of the peptide vaccine. The 3D structure display, docking, and free energy calculation analysis indicate that the binding affinity between the vaccine peptide and Toll-like receptor 3 is high, and the vaccine receptor complex is highly stable. Notably, the vaccine we designed is obtained from the protective protein of the vaccinia and combined with preventive measures to avoid side effects. This vaccine is highly likely to produce an effective and safe immune response against the variola virus infection in the body. IMPORTANCE: In this work, we designed a vaccine with a cluster of multiple T-cell/B-cell epitopes, which should be effective in inducing systematic immune responses against variola virus infection. Besides, this work also provides a reference in vaccine design for preventing monkeypox virus infection, which is currently prevalent.
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Biología Computacional , Epítopos de Linfocito B , Epítopos de Linfocito T , Vacuna contra Viruela , Viruela , Vacunas de Subunidad , Virus de la Viruela , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/química , Vacunas de Subunidad/genética , Humanos , Vacuna contra Viruela/inmunología , Virus de la Viruela/inmunología , Virus de la Viruela/genética , Viruela/prevención & control , Viruela/inmunología , Linfocitos T/inmunología , Linfocitos B/inmunología , Simulación del Acoplamiento Molecular , Péptidos/inmunología , Péptidos/química , InmunoinformáticaRESUMEN
Objective: Rheumatoid arthritis (RA) is a systemic disease that attacks the joints and causes a heavy economic burden on humans worldwide. T cells regulate RA progression and are considered crucial targets for therapy. Therefore, we aimed to integrate multiple datasets to explore the mechanisms of RA. Moreover, we established a T cell-related diagnostic model to provide a new method for RA immunotherapy. Methods: scRNA-seq and bulk-seq datasets for RA were obtained from the Gene Expression Omnibus (GEO) database. Various methods were used to analyze and characterize the T cell heterogeneity of RA. Using Mendelian randomization (MR) and expression quantitative trait loci (eQTL), we screened for potential pathogenic T cell marker genes in RA. Subsequently, we selected an optimal machine learning approach by comparing the nine types of machine learning in predicting RA to identify T cell-related diagnostic features to construct a nomogram model. Patients with RA were divided into different T cell-related clusters using the consensus clustering method. Finally, we performed immune cell infiltration and clinical correlation analyses of T cell-related diagnostic features. Results: By analyzing the scRNA-seq dataset, we obtained 10,211 cells that were annotated into 7 different subtypes based on specific marker genes. By integrating the eQTL from blood and RA GWAS, combined with XGB machine learning, we identified a total of 8 T cell-related diagnostic features (MIER1, PPP1CB, ICOS, GADD45A, CD3D, SLFN5, PIP4K2A, and IL6ST). Consensus clustering analysis showed that RA could be classified into two different T-cell patterns (Cluster 1 and Cluster 2), with Cluster 2 having a higher T-cell score than Cluster 1. The two clusters involved different pathways and had different immune cell infiltration states. There was no difference in age or sex between the two different T cell patterns. In addition, ICOS and IL6ST were negatively correlated with age in RA patients. Conclusion: Our findings elucidate the heterogeneity of T cells in RA and the communication role of these cells in an RA immune microenvironment. The construction of T cell-related diagnostic models provides a resource for guiding RA immunotherapeutic strategies.
Asunto(s)
Artritis Reumatoide , Análisis de la Aleatorización Mendeliana , Sitios de Carácter Cuantitativo , RNA-Seq , Análisis de la Célula Individual , Humanos , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/diagnóstico , Análisis de la Célula Individual/métodos , Nomogramas , Aprendizaje Automático , Linfocitos T/inmunología , Linfocitos T/metabolismo , Perfilación de la Expresión Génica , Análisis de Expresión Génica de una Sola CélulaRESUMEN
We report here the complete genomic sequence of the Chinese bluetongue virus serotype 1 (BTV1) strain SZ97/1. This work is the first to document the complete genomic sequence of a BTV1 strain from China and represents the second complete sequence of BTV1 in the world. The sequence information provided here will help determine the geographic origin of Chinese BTV1 and provide data to facilitate future analyses of the genetic diversity and phylogenetic relationships of BTV strains.
Asunto(s)
Virus de la Lengua Azul/genética , Virus de la Lengua Azul/aislamiento & purificación , Lengua Azul/virología , Genoma Viral , Animales , Virus de la Lengua Azul/clasificación , China , Genómica , Datos de Secuencia Molecular , OvinosRESUMEN
The VP2 protein of bluetongue virus (BTV) is an important structural protein and is the principal antigen responsible for BTV serotype specificity. In this study, we mapped the reactivity of two BTV16-specific monoclonal antibodies (MAbs) and identified two novel serotype-specific linear B cell epitopes on the BTV16 VP2 protein. By screening a series of peptides derived from the BTV16 VP2 protein and expressed as mannose-binding protein fusions, we determined that the linear epitopes recognized by the VP2-specific MAbs 3 G10 and 2B4 were located within the peptides 34EWSGHDVTEIPNRRMF49 and 540KNEDPYVKRTVKPIRA555, respectively. To define the minimal region required for antibody binding within these peptide regions, a series of progressively shorter peptides were synthesized and evaluated for 3 G10 and 2B4 binding. This work defined the motifs 34EWSGHDVTEIPNRRMF49 and 543DPYVKRTVK555 as the minimal linear peptides required for 3 G10 and 2B4 binding, respectively. Alignment of amino acid sequences from a number of BTV16 strains isolated from different regions indicated that these two epitopes are highly conserved among BTV16 strains. Furthermore, these two epitopes are not conserved among other BTV serotypes or prototype members of the genus Orbivirus in the family Reoviridae, as shown by sequence alignments. The MAb reagents and linear epitopes defined here provide the basis for the development of epitope-based serotype-specific differential diagnostic tools and may be useful in the design of epitope-based vaccines.