ABSTRACT
African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.
Subject(s)
African Swine Fever Virus , African Swine Fever , CRISPR-Cas Systems , Endosomes , Membrane Proteins , Virus Internalization , Virus Replication , Animals , Swine , African Swine Fever Virus/genetics , African Swine Fever Virus/physiology , African Swine Fever/virology , African Swine Fever/metabolism , African Swine Fever/genetics , Endosomes/metabolism , Endosomes/virology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Gene Knockout TechniquesABSTRACT
African swine fever is a fatal infectious disease caused by African swine fever virus (ASFV). The high mortality caused by this infectious disease is a significant challenge to the swine industry worldwide. ASFV virulence is related to its ability to antagonize IFN response, yet the mechanism of antagonism is not understood. Recently, a less virulent recombinant virus has emerged that has a EP402R gene deletion within the parental ASFV HLJ/18 (ASFV-ΔEP402R) strain. EP402R gene encodes CD2v. Hence we hypothesized that ASFV uses CD2v protein to evade type I IFN-mediated innate immune response. We found that ASFV-ΔEP402R infection induced higher type I IFN response and increased the expression of IFN-stimulated genes in porcine alveolar macrophages when compared with parental ASFV HLJ/18. Consistent with these results, CD2v overexpression inhibited type I IFN production and IFN-stimulated gene expression. Mechanistically, CD2v, by interacting with the transmembrane domain of stimulator of IFN genes (STING), prevented the transport of STING to the Golgi apparatus, and thereby inhibited the cGMP-AMP synthase-STING signaling pathway. Furthermore, ASFV CD2v disrupted IFNAR1-TYK2 and IFNAR2-JAK1 interactions, and thereby inhibited JAK-STAT activation by IFN-α. In vivo, specific pathogen-free pigs infected with the mutant ASFV-ΔEP402R strain survived better than animals infected with the parental ASFV HLJ/18 strain. Consistent with this finding, IFN-ß protein levels in the peripheral blood of ASFV-ΔEP402R-challenged pigs were significantly higher than in the blood of ASFV HLJ/18-challenged pigs. Taken together, our findings suggest a molecular mechanism in which CD2v inhibits cGMP-AMP synthase-STING and IFN signaling pathways to evade the innate immune response rendering ASFV infection fatal in pigs.
Subject(s)
African Swine Fever Virus , African Swine Fever , Interferon Type I , Swine , Animals , African Swine Fever Virus/genetics , Viral Proteins , Signal Transduction , Gene Expression , Interferon Type I/metabolismABSTRACT
Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.
Subject(s)
Capsid Proteins/immunology , Circovirus/immunology , Animals , Antibodies, Monoclonal , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Circoviridae Infections/virology , Circovirus/metabolism , Circovirus/ultrastructure , Cryoelectron Microscopy , Epitopes , Genotype , Protein Conformation , Swine , Swine Diseases/virologyABSTRACT
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.
Subject(s)
Hemorrhagic Disease Virus, Epizootic/genetics , Reverse Genetics/methods , Virology/methods , Animals , Cell Line , DNA, Complementary/genetics , Hemorrhagic Disease Virus, Epizootic/growth & development , Mesocricetus , Plasmids , RNA, Viral/genetics , Recombination, Genetic , Reoviridae Infections/virologyABSTRACT
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.
Subject(s)
Bluetongue virus/isolation & purification , Polymerase Chain Reaction/methods , Reverse Genetics/methods , Virology/methods , Animals , Bluetongue virus/genetics , Cell Line , Cricetinae , Microbial Viability , Plasmids , TransfectionABSTRACT
The variant strains of porcine epidemic diarrhea virus (PEDV) severely threaten the pig industry worldwide and cause up to 100% mortality in suckling piglets. It is critically important and urgent to develop tools for detection of PEDV infection. In this study, we developed six monoclonal antibodies (mAbs) targeting N protein of PEDV and analyzed their applications on enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), western blot assay, and flow cytometry assay. The results demonstrated that all these six mAbs were IgG1 isotype and κ chain. Among these six mAbs, 3F12 recognizes a linear epitope (VAAVKDALKSLGI) while the other five mAbs recognize different conformational epitopes formed by a specific peptide fragment or the full length of N protein. The functional analysis showed that all these six mAbs were applicable to ELISA, western blot, IFA, and flow cytometry assay. In conclusion, we developed six mAbs against PEDV-N protein to facilitate the early detection of PEDV infection using ELISA, western blot, IFA, and flow cytometry.
Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Viral/analysis , Coronavirus Infections/veterinary , Nucleocapsid/analysis , Porcine epidemic diarrhea virus/isolation & purification , Swine Diseases/virology , Animals , Antibodies, Viral/immunology , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique, Indirect , Immunization , Mice, Inbred BALB C , Nucleocapsid/immunology , Porcine epidemic diarrhea virus/immunology , Swine , Swine Diseases/diagnosisABSTRACT
BACKGROUND: West Nile virus (WNV) is an emerging zoonotic pathogen which is harmful to human and animal health. Effective vaccination in susceptible hosts should protect against WNV infection and significantly reduce viral transmission between animals and from animals to humans. A versatile vaccine suitable for different species that can be delivered via flexible routes remains an essential unmet medical need. In this study, we developed a recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing WNV premembrane/envelope (PrM/E) proteins (designated rLa-WNV-PrM/E) and evaluated its immunogenicity in mice, horses, chickens, ducks and geese. RESULTS: Mouse immunization experiments disclosed that rLa-WNV-PrM/E induces significant levels of WNV-neutralizing antibodies and E protein-specific CD4+ and CD8+ T-cell responses. Moreover, recombinant rLa-WNV-PrM/E elicited significant levels of WNV-specific IgG in horses upon delivery via intramuscular immunization, and in chickens, ducks and geese via intramuscular, oral or intranasal immunization. CONCLUSIONS: Our results collectively support the utility of rLa-WNV-PrM/E as a promising WNV veterinary vaccine candidate for mammals and poultry.
Subject(s)
Mammals/immunology , Newcastle disease virus/genetics , Poultry Diseases/immunology , Viral Vaccines/immunology , West Nile Fever/prevention & control , West Nile virus/immunology , Animals , Antibodies, Viral/immunology , Chickens , Female , Genetic Vectors/genetics , Genetic Vectors/immunology , Humans , Mammals/virology , Mice , Newcastle disease virus/metabolism , Poultry Diseases/prevention & control , Poultry Diseases/virology , Vaccination , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/genetics , West Nile Fever/immunology , West Nile Fever/virology , West Nile virus/geneticsABSTRACT
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.
Subject(s)
Autophagy , Bluetongue virus/physiology , Energy Metabolism , Virus Replication , Animals , Bluetongue virus/pathogenicity , Cells, Cultured , CricetinaeABSTRACT
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.
Subject(s)
Cattle Diseases/virology , Gene Duplication , Gene Rearrangement , Hemorrhagic Disease Virus, Epizootic/genetics , Reoviridae Infections/veterinary , Reverse Genetics/methods , Animals , Cattle , Hemorrhagic Disease Virus, Epizootic/isolation & purification , Hemorrhagic Disease Virus, Epizootic/physiology , Phylogeny , Reoviridae Infections/virology , Virus ReplicationABSTRACT
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.
Subject(s)
Bluetongue virus/pathogenicity , Eukaryotic Initiation Factor-2/metabolism , eIF-2 Kinase/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Autophagy , Bluetongue virus/genetics , Bluetongue virus/physiology , Capsid Proteins/genetics , Capsid Proteins/physiology , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress , Eukaryotic Initiation Factor-2/antagonists & inhibitors , Eukaryotic Initiation Factor-2/genetics , Gene Knockdown Techniques , Heat-Shock Proteins/metabolism , Indoles/pharmacology , Models, Biological , Signal Transduction , Unfolded Protein Response , Virus Replication , eIF-2 Kinase/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Bluetongue virus/immunology , Epitope Mapping , Epitopes, B-Lymphocyte/immunology , Viral Nonstructural Proteins/immunology , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , Conserved Sequence , DNA Mutational AnalysisABSTRACT
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.
Subject(s)
Bluetongue virus/classification , Bluetongue/virology , Genotype , Genotyping Techniques/methods , Oligonucleotide Probes , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Bluetongue virus/genetics , Sensitivity and Specificity , Serogroup , SheepABSTRACT
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.
Subject(s)
Bluetongue virus/immunology , Bluetongue/prevention & control , Immunization Schedule , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Bluetongue/immunology , Bluetongue virus/genetics , Drug Carriers , Fowlpox virus/genetics , Mice , Sheep , Treatment Outcome , Vaccines, DNA/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Vaccines/geneticsABSTRACT
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.
Subject(s)
Cloning, Molecular/methods , Escherichia coli/metabolism , Recombinant Fusion Proteins/metabolism , 3C Viral Proteases , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Escherichia coli/genetics , Green Fluorescent Proteins/genetics , Recombinant Fusion Proteins/genetics , Solubility , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The exploration into nanomaterial-based nonenzymatic biosensors with superb performance in terms of good sensitivity and anti-interference ability in disease marker monitoring has always attained undoubted priority in sensing systems. In this work, we report the design and synthesis of a highly active nanocatalyst, i.e., palladium and platinum nanoparticles (Pt&Pd-NPs) decorated ultrathin nanoporous gold (NPG) film, which is modified on a homemade graphene paper (GP) to develop a high-performance freestanding and flexible nanohybrid electrode. Owing to the structural characteristics the robust GP electrode substrate, and high electrochemically catalytic activities and durability of the permeable NPG support and ultrafine and high-density Pt&Pd-NPs on it, the resultant Pt&Pd-NPs-NPG/GP electrode exhibits excellent sensing performance of low detection limitation, high sensitivity and anti-interference capability, good reproducibility and long-term stability for the detection of small molecular biomarkers hydrogen peroxide (H2O2) and glucose (Glu), and has been applied to the monitoring of H2O2 in different types of live cells and Glu in body fluids such as urine and fingertip blood, which is of great significance for the clinical diagnosis and prognosis in point-of-care testing.
Subject(s)
Biomarkers , Biosensing Techniques , Electrochemical Techniques , Gold , Graphite , Metal Nanoparticles , Palladium , Platinum , Graphite/chemistry , Gold/chemistry , Platinum/chemistry , Palladium/chemistry , Metal Nanoparticles/chemistry , Biomarkers/urine , Humans , Hydrogen Peroxide , Alloys/chemistry , Glucose/analysis , Electrodes , PaperABSTRACT
Genetic changes have occurred in the genomes of prevalent African swine fever viruses (ASFVs) in the field in China, which may change their antigenic properties and result in immune escape. There is usually poor cross-protection between heterogonous isolates, and, therefore, it is important to test the cross-protection of the live attenuated ASFV vaccines against current prevalent heterogonous isolates. In this study, we evaluated the protective efficacy of the ASFV vaccine candidate HLJ/18-7GD against emerging isolates. HLJ/18-7GD provided protection against a highly virulent variant and a lower lethal isolate, both derived from genotype II Georgia07-like ASFV and isolated in 2020. HLJ/18-7GD vaccination prevented pigs from developing ASF-specific clinical signs and death, decreased viral shedding via the oral and rectal routes, and suppressed viral replication after challenges. However, HLJ/18-7GD vaccination did not provide solid cross-protection against genotype I NH/P68-like ASFV challenge in pigs. HLJ/18-7GD vaccination thus shows great promise as an alternative strategy for preventing and controlling genotype II ASFVs, but vaccines providing cross-protection against different ASFV genotypes may be needed in China.
Subject(s)
African Swine Fever Virus , African Swine Fever , Viral Vaccines , Swine , Animals , African Swine Fever/prevention & control , Vaccines, Attenuated/genetics , Viral Proteins/genetics , Genotype , Viral Vaccines/geneticsABSTRACT
African swine fever virus (ASFV) severely threatens the global economy and food security. ASFV encodes >150 genes, but the functions of most of them have yet to be characterized in detail. Here we explored the function of the ASFV CP312R gene and found that CP312R plays an essential role in ASFV replication. Knockout of the CP312R gene terminated viral replication and CP312R knockdown substantially suppressed ASFV infection in vitro. Furthermore, we resolved the crystal structure of pCP312R to 2.3 Å resolution and found that pCP312R has the potential to bind nucleic acids. LC-MS analysis and co-immunoprecipitation assay revealed that pCP312R interacts with RPS27A, a component of the 40S ribosomal subunit. Confocal microscopy showed the interaction between pCP312R and RPS27A leaded to a modification in the subcellular localization of this host protein, which suppresses host protein translation. Renilla-Glo luciferase assay and Ribopuromycylation analysis evidenced that knockout of RPS27A completely aborted the shutoff activity of pCP312R, and trans-complementation of RPS27A recovered pCP312R shutoff activity in RPS27A-knockout cells. Our findings shed light on the function of ASFV CP312R gene in virus infection, which triggers inhibition of host protein synthesis.
Subject(s)
African Swine Fever Virus , Protein Biosynthesis , Ribosomal Proteins , Viral Proteins , Virus Replication , African Swine Fever Virus/metabolism , African Swine Fever Virus/genetics , Animals , Ribosomal Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/chemistry , Viral Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/chemistry , Swine , Host-Pathogen Interactions , Protein Binding , Chlorocebus aethiops , Vero Cells , African Swine Fever/virology , African Swine Fever/metabolismABSTRACT
In order to further reduce the energy consumption of the conventional thermal catalytic oxidation system and improve the degradation efficiency of pollutants, photothermal synergistic catalytic oxidation (PTSCO) system was constructed in this paper with propane as simulated pollutant representing VOCs, and then the modified α-MnO2 catalysts were prepared by using the acid activation method, which were used for the catalytic oxidation of propane in PTSCO. The α-MnO2 with appropriate acid concentration possessed excellent low-temperature reducibility, abundant active oxygen species, fast oxygen migration rate and a large number of acid sites. The optimal catalyst, H0.05-MnO2, had a T90 of 204 °C in the PTSCO system, which reduced by more than 30 °C relative to the α-MnO2 (T90 of 235 °C). Moreover, H0.05-MnO2 demonstrated excellent water resistance and long-term stability (T = 45 h). It was shown that the combination of photocatalysis and thermocatalysis can improve propane degradation by examining the kinetics of propane degradation in the PTSCO system and the conformational relationship of propane degradation by catalysts. Furthermore, a multi-pathway synergistic mechanism between photocatalysis and thermocatalysis in the PTSCO system was proposed. This work provided a theoretical basis for the preparation of high-performance catalysts and the catalytic degradation of propane.
ABSTRACT
Owing to their superior charge retaining and transport characteristics, 2D transition metal dichalcogenides are investigated for practical applications in various memory-cell structures. Herein, we fabricated a quasi-one-terminal 2D memory cell by partially depositing a WSe2 monolayer on an Au electrode, which can be manipulated to achieve efficient charge injection upon the application or removal of external bias. Furthermore, the amount of charge carriers stored in the memory cell could be optically probed because of its close correlation with the fluorescence efficiency of WSe2, allowing us to achieve an electron retention time of â¼300 s at the cryogenic temperature of 4 K. Accordingly, the simplified device structure and the non-contact optical readout of the stored charge carriers present new research opportunities for 2D memory cells in terms of both fundamental mechanism studies and practical development for integrated nanophotonic devices.
ABSTRACT
Due to the absence of effective vaccine and treatment, African swine fever virus (ASFV) control is entirely dependent on accurate and early diagnosis, along with culling of infected pigs. The B646L/p72 is the major capsid protein of ASFV and is an important target for developing a diagnostic assays and vaccines. Herein, we generated a monoclonal antibody (mAb) (designated as 2F11) against the trimeric p72 protein, and a blocking ELISA (bELISA) was established for the detection of both genotype I and II ASFV antibodies. To evaluate the performance of the diagnostic test, a total of 506 porcine serum samples were tested. The average value of percent of inhibition (PI) of 133 negative pig serum was 8.4 % with standard deviation (SD) 6.5 %. Accordingly, the cut-off value of the newly established method was set at 28 % (mean + 3SD). Similarly, a receiver operating characteristic (ROC) was applied to determine the cut off value and the p72-bELISA exhibited a sensitivity of 100 % and a specificity of 99.33 % when the detection threshold was set at 28 %. The bELISA was also able to specifically recognize anti-ASFV sera without cross-reacting with other positive serums for other major swine pathogens. Moreover, by designing a series of overlapped p72 truncated proteins, the linear B cell epitope recognized by 2F11 mAb was defined to be 283NSHNIQ288. Amino acid sequence comparison revealed that the amino acid sequence 283NSHNIQ288 is highly conserved between different ASFV isolates. Our findings indicate that the newly established mAb based blocking ELISA may have a great potential in improving the detection of ASFV antibodies and provides solid foundation for further studies.