ABSTRACT
Japanese encephalitis virus (JEV), a member of the Flaviviridae family and a flavivirus, is known to induce acute encephalitis. Vimentin protein has been identified as a potential receptor for JEV, engaging in interactions with the viral membrane protein. The Fc fragment, an integral constituent of immunoglobulins, plays a crucial role in antigen recognition by dendritic cells (DCs) or phagocytes, leading to subsequent antigen presentation, cytotoxicity, or phagocytosis. In this study, we fused the receptor of JEV vimentin with the Fc fragment of IgG and expressed the resulting vimentin-Fc fusion protein in Escherichia coli. Pull-down experiments demonstrated the binding ability of the vimentin-Fc fusion protein to JEV virion in vitro. Additionally, we conducted inhibition assays at the cellular level, revealing the ability of vimentin-Fc protein suppressing JEV replication, it may be a promising passive immunotherapy agent for JEV. These findings pave the way for potential therapeutic strategies against JEV.
ABSTRACT
Rabies has been with humans for a long time, and its special transmission route and almost 100 % lethality rate made it once a nightmare for humans. In this study, by predicting the rabies virus glycoprotein outer membrane region and nucleoprotein B-cell antigenic epitopes, the coding sequence of the predicted highly antigenic polypeptide region obtained was assembled using the eukaryotic expression vector pcDNA3.1(-), and then E. coli was used as the delivery vector. The immunogenicity and protective properties of the vaccine were verified by in vivo and in vitro experiments, which demonstrated that the vaccine could produce antibodies in mice and prolong the survival time of mice exposed to the strong virus without any side effects. This study demonstrated that the preparation of an oral rabies DNA vaccine using food-borne microorganisms as a transport vehicle is feasible and could be a new strategy to eradicate rabies starting with wild animals.
Subject(s)
Rabies Vaccines , Rabies virus , Rabies , Vaccines, DNA , Humans , Animals , Mice , Rabies/prevention & control , Escherichia coli , Antibodies, Viral , Rabies Vaccines/genetics , Rabies virus/genetics , Epitopes, B-Lymphocyte/geneticsABSTRACT
Hepatitis B virus (HBV) infection is a global public health problem that is closely related to liver cirrhosis and hepatocellular carcinoma (HCC). The prevalence of acute and chronic HBV infection, liver cirrhosis, and HCC has significantly decreased as a result of the introduction of universal HBV vaccination programs. The first hepatitis B vaccine approved was developed by purifying the hepatitis B surface antigen (HBsAg) from the plasma of asymptomatic HBsAg carriers. Subsequently, recombinant DNA technology led to the development of the recombinant hepatitis B vaccine. Although there are already several licensed vaccines available for HBV infection, continuous research is essential to develop even more effective vaccines. Prophylactic hepatitis B vaccination has been important in the prevention of hepatitis B because it has effectively produced protective immunity against hepatitis B viral infection. Prophylactic vaccines only need to provoke neutralizing antibodies directed against the HBV envelop proteins, whereas therapeutic vaccines are most likely needed to induce a comprehensive T cell response and thus, should include other HBV antigens, such as HBV core and polymerase. The existing vaccines have proven to be highly effective in preventing HBV infection, but ongoing research aims to improve their efficacy, duration of protection, and accessibility. The routine administration of the HBV vaccine is safe and well-tolerated worldwide. The purpose of this type of immunization is to trigger an immunological response in the host, which will halt HBV replication. The clinical efficacy and safety of the HBV vaccine are affected by a number of immunological and clinical factors. However, this success is now in jeopardy due to the breakthrough infections caused by HBV variants with mutations in the S gene, high viral loads, and virus-induced immunosuppression. In this review, we describe various types of available HBV vaccines, along with the recent progress in the ongoing battle to develop new vaccines against HBV.
ABSTRACT
More than 250 million people worldwide have chronic hepatitis B virus (HBV) infections, resulting in over 1 million annual fatalities because HBV cannot be adequately treated with current antivirals. Hepatocellular carcinoma (HCC) risk is elevated in the presence of the HBV. Novel and powerful medications that specifically target the persistent viral components are needed to remove infection. This study aimed to use HepG2.2.15 cells and the rAAV-HBV1.3 C57BL/6 mouse model established in our laboratory to examine the effects of 16F16 on HBV. The transcriptome analysis of the samples was performed to examine the impact of 16F16 therapy on host factors. We found that the HBsAg and HBeAg levels significantly decreased in a dose-dependent manner following the 16F16 treatment. 16F16 also showed significant anti-hepatitis B effects in vivo. The transcriptome analysis showed that 16F16 regulated the expression of several proteins in HBV-producing HepG2.2.15 cells. As one of the differentially expressed genes, the role of S100A3 in the anti-hepatitis B process of 16F16 was further investigated. The expression of the S100A3 protein significantly decreased following the 16F16 therapy. And upregulation of S100A3 caused an upregulation of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells. Similarly, knockdown of S100A3 significantly reduced the levels of HBsAg, HBeAg, and HBV DNA. Our findings proved that S100A3 might be a new target for combating HBV pathogenesis. 16F16 can target several proteins involved in HBV pathogenesis, and may be a promising drug precursor molecule for the treatment of HBV.
Subject(s)
Carcinoma, Hepatocellular , Hepatitis B, Chronic , Liver Neoplasms , Animals , Mice , DNA, Viral/genetics , Gene Expression Profiling , Hepatitis B e Antigens , Hepatitis B Surface Antigens/genetics , Hepatitis B virus , Hepatitis B, Chronic/drug therapy , Mice, Inbred C57BL , Transcriptome , Humans , Hep G2 Cells/metabolism , Hep G2 Cells/virology , Antiviral Agents/pharmacologyABSTRACT
A histidine (His)-tag is composed of six His residues and typically exerts little influence on the structure and solubility of expressed recombinant fusion proteins. Purification methods for recombinant proteins containing His-tags are relatively well-established, thus His-tags are widely used in protein recombination technology. We established a one-step enzyme-linked immunosorbent assay (ELISA) for His-tagged recombinant proteins. We analyzed variable heavy and light chains of the anti-His-tag monoclonal antibody 4C9 and used BLAST analyses to determine variable zones in light (VL) and heavy chains (VH). VH, VL, and alkaline phosphatase (ALP) regions were connected via a linker sequence and ligated into the pGEX-4T-1 expression vector. Different recombinant proteins with His tags were used to evaluate and detect ALP-scFv activity. Antigen and anti-His-scFv-ALP concentrations for direct ELISA were optimized using the checkerboard method. ZIKV-NS1, CHIKV-E2, SCRV-N, and other His-tag fusion proteins demonstrated specific reactions with anti-His-scFv-ALP, which were accurate and reproducible when the antigen concentration was 50 µg mL-1 and the antibody concentration was 6.25 µg mL-1. For competitive ELISA, we observed a good linear relationship when coating concentrations of recombinant human anti-Müllerian hormone (hAMH) were between 0.78 and 12.5 µg mL-1. Our direct ELISA method is simple, rapid, and accurate. The scFv antibody can be purified using a prokaryotic expression system, which provides uniform product quality and reduces variations between batches.
ABSTRACT
Dengue fever is a classic mosquito viral disease. Dengue virus non-structural protein-1 as a membrane-associated homologous dimer anchored to the surface of infected cells and also secreted into the blood. The nonstructural protein-1 levels are related to disease severity, and the presence of nonstructural protein-1 secreted from cells to the serum of people infected with the dengue virus is an early marker of infection. Paired antibodies are key in the establishment of rapid detection technology. In this study, the prepared recombinant nonstructural protein-1 protein of dengue virus serotype 3 was purified by the prokaryotic expression, and prepared monoclonal antibodies by cell fusion. A method for paired antibody screening was established based on the N-hydroxy succinimide-nanobeads and the prepared monoclonal antibodies. A simple and rapid point-of-care system integrating the paired antibodies and lateral flow assay was established to verify the screened antibody pairs. The results confirmed that the antibody pair screening method based on N-hydroxy succinimide-nanobeads is feasible.
Subject(s)
Dengue Virus , Dengue , Animals , Antibodies, Viral , Dengue/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Viral Nonstructural ProteinsABSTRACT
The catalysis of disulphide (SS) bonds is the most important characteristic of protein disulphide isomerase (PDI) family. Catalysis occurs in the endoplasmic reticulum, which contains many proteins, most of which are secretory in nature and that have at least one s-s bond. Protein disulphide isomerase A3 (PDIA3) is a member of the PDI family that acts as a chaperone. PDIA3 is highly expressed in response to cellular stress, and also intercept the apoptotic cellular death related to endoplasmic reticulum (ER) stress, and protein misfolding. PDIA3 expression is elevated in almost 70% of cancers and its expression has been linked with overall low cell invasiveness, survival and metastasis. Viral diseases present a significant public health threat. The presence of PDIA3 on the cell surface helps different viruses to enter the cells and also helps in replication. Therefore, inhibitors of PDIA3 have great potential to interfere with viral infections. In this review, we summarize what is known about the basic structure, functions and role of PDIA3 in viral infections. The review will inspire studies of pathogenic mechanisms and drug targeting to counter viral diseases.
Subject(s)
Protein Disulfide-Isomerases/metabolism , Virus Diseases/enzymology , Virus Diseases/virology , Virus Internalization , Virus Replication , Viruses/growth & development , Animals , Antiviral Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Host-Pathogen Interactions , Humans , Protein Disulfide-Isomerases/antagonists & inhibitors , Virus Diseases/drug therapy , Viruses/pathogenicityABSTRACT
Rabies virus is a highly neurophilic negative-strand RNA virus with high lethality and remains a huge public health problem in developing countries to date. The double-stranded RNA-binding protein Staufen1 (STAU1) has multiple functions in RNA virus replication, transcription, and translation. However, its function in RABV infection and its mechanism of action are not clear. In this study, we investigated the role of host factor STAU1 in RABV infection of SH-SY-5Y cells. Immunofluorescence, TCID50 titers, confocal microscopy, quantitative real-time PCR and Western blotting were carried out to determine the molecular function and subcellular distribution of STAU1 in these cell lines. Expression of STAU1 in SH-SY-5Y cells was down-regulated by RNA interference or up-regulated by transfection of eukaryotic expression vectors. The results showed that N proficiently colocalized with STAU1 in SH-SY-5Y at 36 h post-infection, and the expression level of STAU1 was also proportional to the time of infection. Down-regulation of STAU1 expression increased the number of Negri body-like structures, enhanced viral replication, and a caused 10-fold increase in viral titers. Meanwhile, N protein and G protein mRNA levels also accumulated gradually with increasing infection time, which implied that STAU1 inhibited rabies virus infection of SH-SY-5Y cells in vitro. In conclusion, our results provide important clues for the detailed replication mechanism of rabies virus and the discovery of therapeutic targets.
Subject(s)
Cytoskeletal Proteins/metabolism , RNA-Binding Proteins/metabolism , Rabies virus/physiology , Virus Replication , Cell Line, Tumor , Cytoskeletal Proteins/genetics , Host-Pathogen Interactions , Humans , Inclusion Bodies, Viral/metabolism , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , RNA Interference , RNA-Binding Proteins/genetics , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolismABSTRACT
DEAD-box (DDX) helicases are critical for recognizing viral nucleic acids to regulate antiviral innate immunity. Although DDX5 has been reported to participate in various virus infection, whether DDX5 regulates innate immune responses and its underlying mechanisms are still unknown. Here, we report that DDX5 is a negative regulator of type I IFN (IFN-I) production in antiviral responses. DDX5 knockdown significantly promoted DNA or RNA virus infection-induced IFN-I production and IFN-stimulated genes (ISGs) expression, while ectopic expression of DDX5 inhibited IFN-I production and promoted viral replication. Furthermore, we found that DDX5 specifically interacted with serine/threonine-protein phosphatase 2 A catalytic subunit beta (PP2A-Cß) and viral infection enhanced the interaction between DDX5 and PP2A-Cß. Besides, PP2A-Cß interacted with IFN regulatory factor 3 (IRF3), and PP2A-Cß knockdown promoted viral infection-induced IRF3 phosphorylation and IFN-I production. In addition, DDX5 knockdown rendered the mice more resistant to viral infection and enhanced antiviral innate immunity in vivo. Thus, DDX5 suppresses IFN-I antiviral innate immune response by interacting with PP2A-Cß to deactivate IRF3. Together, these findings identify a negative role of DDX5 on regulating IFN-I signaling in innate immune responses.
Subject(s)
DEAD-box RNA Helicases/metabolism , Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Interferon Type I/metabolism , Protein Phosphatase 2/metabolism , Animals , Female , Gene Knockdown Techniques , Mice, Inbred C57BL , Phosphorylation , Protein Binding , Up-Regulation/genetics , Virus Diseases/metabolism , Virus Diseases/pathologyABSTRACT
The use of exogenous functional microorganisms to regulate biogenic amine (BA) content is a common approach in fermentation systems. Here, to better understand the microbial traits of succession trajectories in resource-based and biotic interference systems, the BA-related primary and secondary succession were tracked during industrial semidry Chinese rice wine (CRW) fermentation. Dominant abundance and BA-associated microbial functionality based on phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) indicated that Citrobacter, Acinetobacter, Lactobacillus, Exiguobacterium, Bacillus, Pseudomonas, and Enterobacter spp. prominently contributed to the decarboxylase gene family in CRW. The expression levels of tyrosine decarboxylase (tyrDC), ornithine decarboxylase (odc), and agmatine deiminase (aguA) genes were assessed by quantitative PCR (qPCR). The transcription levels of these genes did not correlate with the BA formation rate during postfermentation, indicating that acidification and carbon source depletion upregulated the expression and microbes launch the dormancy strategy to respond to unfavorable conditions. Furthermore, microbial interference with CRW fermentation by Lactobacillus plantarum (ACBC271) and Staphylococcus xylosus (CGMCC1.8382) coinoculated at a ratio of 1:2 exhibited the best synergetic control of BA content. Spearman correlations revealed that Lactobacillus and Staphylococcus exhibited influence on BA-associated microbiota (|ρ| > 0), Exiguobacterium and Pseudomonas were strongly suppressed by Lactobacillus (ρ = -0.867 and ρ = -0.782, respectively; P < 0.05), and Staphylococcus showed the strongest inhibitory effect toward Lactobacillus (ρ = -0.115) and Citrobacter (ρ = -0.188) in the coinoculated 1:2 group. The high inhibitory effect of exogenous added strains on specific bacteria presented evidence for the obtained BA-associated contributors. Overall, this work provides important insight into the microbial traits that rely on resource usage and functional microbiota within food microbial ecology.IMPORTANCE Understanding the shifting patterns of substance usage and microbial interactions is a fundamental objective within microbiology and ecology. Analyses of primary and secondary microbial succession allow for determinations of taxonomic diversity, community traits, and functional transformations over time or after a disturbance. The kinetics of BA generation and the patterns of resource consumption, functional metagenome prediction, and microbial interactions were profiled to elucidate the equilibrium mechanism of microbial systems. Secondary succession after a disturbance triggers a change in resource usage, which in turn affects primary succession and metabolism. In this study, the functional potential of exogenous microorganisms under disturbance synergized with secondary succession strategies, including rebalancing and dormancy, which ultimately reduced BA accumulation. Thus, this succession system could facilitate the settling of essential issues with respect to microbial traits that rely on resource usage and microbial interactions that occur in natural ecosystems.