ABSTRACT
Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.
Subject(s)
Carrier Proteins , Comovirus , Nicotiana , Plant Diseases , Plant Proteins , Vigna , Comovirus/metabolism , Comovirus/physiology , Comovirus/genetics , Vigna/virology , Vigna/metabolism , Nicotiana/virology , Nicotiana/metabolism , Nicotiana/genetics , Carrier Proteins/metabolism , Carrier Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/genetics , Plant Diseases/virology , Cysteine Proteases/metabolism , Cysteine Proteases/genetics , Plants, Genetically Modified , Viral Proteins/metabolism , Viral Proteins/genetics , Capsid Proteins/metabolism , Capsid Proteins/genetics , Potyvirus/physiology , Potyvirus/metabolism , EndopeptidasesABSTRACT
Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.
Subject(s)
Capsid Proteins , Capsid , Dependovirus , Genetic Vectors , Liver , Dependovirus/genetics , Animals , Humans , Mice , Genetic Vectors/genetics , Capsid/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Liver/metabolism , Transduction, Genetic , Gene Transfer Techniques , Machine Learning , Genetic Therapy/methods , Macaca , Hepatocytes/metabolism , HEK293 Cells , Genetic Engineering/methodsABSTRACT
The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing "empty" capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.
Subject(s)
Dependovirus , Dependovirus/genetics , Dependovirus/isolation & purification , Chromatography, Ion Exchange/methods , Humans , Capsid/chemistry , Capsid Proteins/genetics , Capsid Proteins/chemistry , Capsid Proteins/isolation & purification , Capsid Proteins/analysis , HEK293 CellsABSTRACT
The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting â¼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.
Subject(s)
Bacteriophage M13 , Metals, Rare Earth , Metals, Rare Earth/chemistry , Metals, Rare Earth/isolation & purification , Bacteriophage M13/chemistry , Bacteriophage M13/genetics , Lanthanoid Series Elements/chemistry , Capsid Proteins/chemistry , Capsid Proteins/isolation & purification , Capsid Proteins/genetics , Peptides/chemistry , Hydrogen-Ion ConcentrationABSTRACT
Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.
Subject(s)
Adaptor Protein Complex 1 , Capsid Proteins , Hepatitis E virus , Hepatitis E virus/metabolism , Hepatitis E virus/physiology , Hepatitis E virus/genetics , Humans , Adaptor Protein Complex 1/metabolism , Adaptor Protein Complex 1/genetics , Capsid Proteins/metabolism , Capsid Proteins/genetics , Protein Transport , Viral Proteins/metabolism , Viral Proteins/genetics , Virus Assembly , Hepatitis E/metabolism , Hepatitis E/virologyABSTRACT
The unenveloped Bluetongue virus capsid comprises several structural layers, the inner two comprising a core, which assembles before addition of the outer proteins, VP2 and VP5. Two symmetric trimers of VP5 fit like pegs into two distinct pits on the core and undergo pH conformational changes in the context of the virus, associated with cell entry. Here we show that in isolation VP5 alone undergoes essentially the same changes with pH and confirm a helical transition, indicating that VP5 is a motor during cell entry. In the absence of VP5 the two pits on the core differ from each other, presumably due to the asymmetric underlying structure of VP3, the innermost capsid protein. On insertion of VP5 these pits become closely similar and remain similar at low pH whilst VP5 is present. This natural asymmetry presumably destabilises the attachment of VP5, facilitating ejection upon low pH, membrane penetration and cell entry.
Subject(s)
Bluetongue virus , Capsid Proteins , Bluetongue virus/physiology , Bluetongue virus/chemistry , Hydrogen-Ion Concentration , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Capsid Proteins/genetics , Virus Internalization , Animals , Protein ConformationABSTRACT
Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6-10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3-4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 1010 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity.
Subject(s)
Bone Marrow Cells , Circovirus , Animals , Swine , Circovirus/physiology , Circovirus/isolation & purification , Circovirus/genetics , Bone Marrow Cells/virology , Cells, Cultured , Circoviridae Infections/virology , Circoviridae Infections/veterinary , Swine Diseases/virology , Capsid Proteins/genetics , Capsid Proteins/metabolism , Virus Cultivation/methodsABSTRACT
Nervous necrosis virus (NNV) capsid protein plays an important role in producing viral particles without any genetic elements. Thus, NNV is a promising candidate for vaccine development and is widely used for constructing vaccines, including DNA, recombinant proteins, and virus-like particles (VLPs). Our study aimed to investigate the potential of NNV capsid protein (NNV) and NNV capsid protein fused to enhanced green fluorescent protein (NNV-EGFP) through VLP formation and whether their application can induce specific antibody responses against certain antigens. We focused on producing DNA and recombinant protein vaccines consisting of the genes for NNV, EGFP, and NNV-EGFP. The approach using NNV-EGFP allowed NNV to act as a carrier or inducer while EGFP was incorporated as part of the capsid protein, thereby enhancing the immune response. In vitro studies demonstrated that all DNA vaccines expressed in HINAE cells resulted in varying protein expression levels, with particularly low levels observed for pNNV and pNNV-EGFP. Consequently, structural proteins derived from HINAE cells could not be observed using transmission electron microscopy (TEM). In contrast, recombinant proteins of NNV and NNV-EGFP were expressed through the Escherichia coli expression system. TEM revealed that rNNV was assembled into VLPs with an approximate size of 30 nm, whereas rNNV-EGFP presented particles ranging from 10 nm to 50 nm in size. For the vaccination test, DNA vaccination marginally induced specific antibody responses in Japanese flounder compared to unvaccinated fish. Meanwhile, NNV and NNV-EGFP recombinant vaccines enhanced a greater anti-NNV antibody response than the others, whereas antibody responses against EGFP were also marginal. These results indicate that NNV capsid protein-based antigens, presenting as particles, play an important role in eliciting a specific anti-NNV antibody response and have the potential to improve fish immune responses.
Subject(s)
Capsid Proteins , Fish Diseases , Nodaviridae , Viral Vaccines , Animals , Nodaviridae/immunology , Capsid Proteins/immunology , Capsid Proteins/genetics , Fish Diseases/immunology , Fish Diseases/prevention & control , Viral Vaccines/immunology , Viral Vaccines/administration & dosage , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , RNA Virus Infections/veterinary , RNA Virus Infections/immunology , RNA Virus Infections/prevention & control , Vaccines, DNA/immunology , Vaccines, DNA/administration & dosage , Vaccine Development , Vaccines, Virus-Like Particle/immunology , Vaccines, Virus-Like Particle/administration & dosageABSTRACT
Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.
Subject(s)
Antigens, Viral , Baculoviridae , Spike Glycoprotein, Coronavirus , Animals , Baculoviridae/genetics , Antigens, Viral/genetics , Antigens, Viral/immunology , Sf9 Cells , Humans , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Recombinant Proteins/genetics , Cell Line , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/immunology , Vaccines, Virus-Like Particle/biosynthesis , Capsid Proteins/genetics , Capsid Proteins/immunology , Glycosylation , Insecta/genetics , Spodoptera , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunologyABSTRACT
Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.
Subject(s)
Dependovirus , Peptides , Dependovirus/genetics , Animals , Peptides/chemistry , Peptides/genetics , Genetic Vectors/genetics , Virion/genetics , Baculoviridae/genetics , Sf9 Cells , Humans , Cell Line , Capsid Proteins/genetics , Capsid Proteins/isolation & purificationABSTRACT
BACKGROUND: Canine parvovirus type 2 (CPV-2) is the most common enteric virus that infects canids. CPV is the causative agent of a contagious disease defined mostly by clinical gastrointestinal signs in dogs. During the late 1970s, CPV-2 emerged as a new virus capable of infecting domestic dogs and growing across the world. The VP2 gene stands out as a key determinant in the pathogenicity, antigenicity, and host interactions of CPV-2. AIMS: The molecular characterization of the VP2 gene is crucial for understanding CPV evolution and epidemiology. MATERIALS & METHODS: Genes encoding the VP2 protein were sequenced and compared to reference strains worldwide. The maximum likelihood method was used to build a phylogenetic tree using CPV VP2 gene nucleotide sequences. RESULTS: Our phylogenetic analysis of the VP2 gene revealed that five strains were very similar and clustered together, and three strains were in the 2b clade, whereas the other two were in the 2a/2b clade. DISCUSSION: This paper reports the molecular characterization of two novel CPV-2a/2b subtypes in dogs with gastrointestinal symptoms. Genetic analysis was conducted on a CPV genomic region encompassing one of the open reading frames (ORFs) encoding the structural protein VP2. Sequence analysis indicates new and unreported sequence changes, mainly affecting the VP2 gene, which includes the mutations Ser297Ala and Leu87Met. This study represents the first evidence of a new CPV-2a/2b subtype in Türkiye. Due to VP2's crucial role in encoding the capsid protein of CPV-2 and its significant involvement in the host-virus interaction, it is critical to closely monitor its evolutionary changes and be cautious while searching for novel or pre-existing subtypes. CONCLUSION: This study highlights the significance of continuous molecular research for acquiring more insights on the circulation of novel CPV mutants.
Subject(s)
Genetic Variation , Parvovirus, Canine , Parvovirus, Canine/classification , Parvovirus, Canine/genetics , Animals , Dogs , Phylogeny , Capsid Proteins/chemistry , Capsid Proteins/genetics , Gastrointestinal Diseases/veterinary , Gastrointestinal Diseases/virology , Parvoviridae Infections/veterinary , Parvoviridae Infections/virology , Turkey , Species Specificity , GenotypeABSTRACT
BACKGROUND: High-risk human papillomavirus (HR-HPV) infection is an important factor for the development of cervical cancer. HPV18 is the second most common HR-HPV after HPV16. METHODS: In this study, MEGA11 software was used to analyze the variation and phylogenetic tree of HPV18 E6-E7 and L1 genes. The selective pressure to E6, E7 and L1 genes was estimated using pamlX. In addition, the B cell epitopes of L1 amino acid sequences and T cell epitopes of E6-E7 amino acid sequences in HPV18 were predicted by ABCpred server and IEDB website, respectively. RESULTS: A total of 9 single nucleotide variants were found in E6-E7 sequences, of which 2 were nonsynonymous variants and 7 were synonymous variants. Twenty single nucleotide variants were identified in L1 sequence, including 11 nonsynonymous variants and 9 synonymous variants. Phylogenetic analysis showed that E6-E7 and L1 sequences were all distributed in A lineage. In HPV18 E6, E7 and L1 sequences, no positively selected site was found. The nonconservative substitution R545C in L1 affected hypothetical B cell epitope. Two nonconservative substitutions, S82A in E6, and R53Q in E7, impacted multiple hypothetical T cell epitopes. CONCLUSION: The sequence variation data of HPV18 may lay a foundation for the virus diagnosis, further study of cervical cancer and vaccine design in central China.
Subject(s)
Genetic Variation , Human papillomavirus 18 , Oncogene Proteins, Viral , Papillomavirus E7 Proteins , Phylogeny , Oncogene Proteins, Viral/genetics , China , Humans , Human papillomavirus 18/genetics , Human papillomavirus 18/classification , Papillomavirus E7 Proteins/genetics , Capsid Proteins/genetics , Female , Epitopes, T-Lymphocyte/genetics , Papillomavirus Infections/virology , Repressor Proteins/genetics , Epitopes, B-Lymphocyte/genetics , DNA-Binding ProteinsABSTRACT
Coxsackievirus A16 (CV-A16) is a significant etiologic agent of hand, foot, and mouth disease (HFMD) and herpangina (HA), with the capacity to progress to severe complications, including encephalitis, aseptic meningitis, acute flaccid paralysis, myocarditis, and other critical conditions. Beijing's epidemiological surveillance system, established in 2008, encompasses 29 hospitals and 16 district disease control centers. From 2019 to 2021, the circulation of CV-A16 was characterized by the co-circulation of B1a and B1b clades. Multiple cases of HFMD linked to clade B1c has not been reported in Beijing until 2022. This study enrolled 400 HFMD and 493 HA cases. Employing real-time RT-PCR, 368 enterovirus-positive cases were identified, with 180 selected for sequencing. CV-A16 was detected in 18.89% (34/180) of the cases, second only to CV-A6, identified in 63.33% (114/180). Full-length VP1 gene sequences were successfully amplified and sequenced in 22 cases, revealing the presence of clades B1a, B1b, and B1c in 14, 3, and 5 cases, respectively. A cluster of five B1c clade cases occurred between June 29 and July 17, 2022, within a 7-km diameter region in Shunyi District. Phylogenetic analysis of five complete VP1 gene sequences and two full-genome sequences revealed close clustering with the 2018 Indian strain (GenBank accession: MH780757.1) within the B1c India branch, with NCBI BLAST results showing over 98% similarity. Comparative sequence analysis identified three unique amino acid variations (P3S, V25A, and I235V). The 2022 Shunyi District HFMD cases represent the first instances of spatiotemporally correlated CV-A16 B1c clade infections in Beijing, underscoring the necessity for heightened surveillance of B1c clade CV-A16 in HFMD and HA in this region.
Subject(s)
Hand, Foot and Mouth Disease , Phylogeny , Humans , Beijing/epidemiology , Hand, Foot and Mouth Disease/virology , Hand, Foot and Mouth Disease/epidemiology , Male , Female , Child, Preschool , Infant , Child , Genotype , Enterovirus/genetics , Enterovirus/classification , Enterovirus/isolation & purification , Capsid Proteins/genetics , Adolescent , Epidemiological MonitoringABSTRACT
Adeno-associated viruses 2 (AAV2) are minute viruses renowned for their capacity to infect human cells and akin organisms. They have recently emerged as prominent candidates in the field of gene therapy, primarily attributed to their inherent non-pathogenic nature in humans and the safety associated with their manipulation. The efficacy of AAV2 as gene therapy vectors hinges on their ability to infiltrate host cells, a phenomenon reliant on their competence to construct a capsid capable of breaching the nucleus of the target cell. To enhance their infection potential, researchers have extensively scrutinized various combinatorial libraries by introducing mutations into the capsid, aiming to boost their effectiveness. The emergence of high-throughput experimental techniques, like deep mutational scanning (DMS), has made it feasible to experimentally assess the fitness of these libraries for their intended purpose. Notably, machine learning is starting to demonstrate its potential in addressing predictions within the mutational landscape from sequence data. In this context, we introduce a biophysically-inspired model designed to predict the viability of genetic variants in DMS experiments. This model is tailored to a specific segment of the CAP region within AAV2's capsid protein. To evaluate its effectiveness, we conduct model training with diverse datasets, each tailored to explore different aspects of the mutational landscape influenced by the selection process. Our assessment of the biophysical model centers on two primary objectives: (i) providing quantitative forecasts for the log-selectivity of variants and (ii) deploying it as a binary classifier to categorize sequences into viable and non-viable classes.
Subject(s)
Mutation , Humans , Capsid Proteins/genetics , Dependovirus/genetics , Parvovirinae/geneticsABSTRACT
The poliovirus (PV) enters the central nervous system (CNS) via the bloodstream, suggesting the existence of a mechanism to cross the blood-brain barrier. Here, we report that PV capsid proteins (VP1 and VP3) can penetrate cells, with VP3 being more invasive. Two independent parts of VP3 are responsible for this function. Both peptides can penetrate human umbilical cord vascular endothelial cells, and one peptide of VP3 could also penetrate peripheral blood mononuclear cells. In an in vitro blood-brain barrier model using rat-derived astrocytes, pericytes, and endothelial cells, both peptides were observed to traverse from the blood side to the brain side at 6 h after administration. These results provide insights into the molecular mechanisms underlying PV invasion into the CNS.
Subject(s)
Blood-Brain Barrier , Capsid Proteins , Poliovirus , Capsid Proteins/metabolism , Capsid Proteins/genetics , Humans , Poliovirus/genetics , Poliovirus/metabolism , Poliovirus/physiology , Animals , Rats , Blood-Brain Barrier/metabolism , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/cytology , Astrocytes/metabolismABSTRACT
Adjuvant is a major supplementary component of vaccines to boost adaptive immune responses. To select an efficient adjuvant from the heat-labile toxin B subunit (LTB) of E. coli, four LTB mutants (numbered LTB26, LTB34, LTB57, and LTB85) were generated by multi-amino acid random replacement. Mice have been intranasally vaccinated with human rotavirus VP8 admixed. Among the four mutants, enzyme-linked immunosorbent assay (ELISA) revealed that LTB26 had enhanced mucosal immune adjuvanticity compared to LTB, showing significantly enhanced immune responses in both serum IgG and mucosal sIgA levels. The 3D modeling analysis suggested that the enhanced immune adjuvanticity of LTB26 might be due to the change of the first LTB α-helix to a ß-sheet. The molecular mechanism was studied using transcriptomic and flow cytometric (FCM) analysis. The transcriptomic data demonstrated that LTB26 enhanced immune response by enhancing B cell receptor (BCR) and major histocompatibility complex (MHC) II+-related pathways. Furthermore, LTB26 promoted Th1 and Th2-type immune responses which were confirmed by detecting IFN-γ and IL-4 expression levels. Immunohistochemical analysis demonstrated that LTB26 enhanced both Th1 and Th2 type immunity. Therefore, LTB26 was a potent mucosal immune adjuvant meeting the requirement for use in human clinics in the future.
Subject(s)
Adjuvants, Immunologic , Enterotoxins , Escherichia coli Proteins , Mice, Inbred BALB C , Animals , Mice , Enterotoxins/immunology , Enterotoxins/genetics , Escherichia coli Proteins/immunology , Escherichia coli Proteins/genetics , Capsid Proteins/immunology , Capsid Proteins/genetics , Bacterial Toxins/immunology , Bacterial Toxins/genetics , Escherichia coli/immunology , Rotavirus/immunology , Immunity, Mucosal/immunology , Antigens, Viral/immunology , Antigens, Viral/genetics , Mutation , Rotavirus Infections/immunology , Female , Rotavirus Vaccines/immunology , Rotavirus Vaccines/administration & dosage , Humans , Immunoglobulin G/immunologyABSTRACT
Human noroviruses (HuNoVs) are highly contagious pathogens responsible of norovirus-associated acute gastroenteritis (AGE). GII.4 is the prevailing HuNoV genotype worldwide. Currently there are no studies on the molecular monitoring and phylogenetic analysis of HuNoVs in the territory of the Sverdlovsk region; therefore, it is not possible to objectively assess their genetic diversity. The aim of the study is to carry out genotyping and phylogenetic analysis of HuNoVs in the Sverdlovsk region from 2022 to 2023. Fecal samples (n = 510) were collected from children suffering from HuNoV-AGE in municipalities of the Sverdlovsk region and the capsid genotype was determined by amplifying the ORF1/ORF2 junction. Of the 196 HuNoVs typed, which represent 38% of the studied samples, the largest share of HuNoV genotypes belong to the GII genogroup-86%, followed by the GI genogroup-14%. Noroviruses GII.4 and GII.17 were the co-dominant capsid genotypes (33.2% each). Phylogenetic analysis demonstrates that the identified sequences on the territory of the Sverdlovsk region have the smallest genetic distance, which gives grounds for their unification into a common cluster. Routine monitoring and phylogenetic analysis of circulating norovirus pathogens spectrum will enable timely tracking of HuNoVs genetic diversity and evolutionary events. This will lead to the development of more effective anti-epidemic measures, ultimately reducing the burden of infectious diseases.
Subject(s)
Caliciviridae Infections , Feces , Gastroenteritis , Genetic Variation , Genotype , Norovirus , Phylogeny , Norovirus/genetics , Norovirus/classification , Norovirus/isolation & purification , Humans , Caliciviridae Infections/virology , Caliciviridae Infections/epidemiology , Russia/epidemiology , Gastroenteritis/virology , Gastroenteritis/epidemiology , Feces/virology , Capsid Proteins/genetics , RNA, Viral/genetics , Child , CitiesABSTRACT
After the first phase of the COVID-19 pandemic in Europe, a new highly pathogenic variant of echovirus 11 (E11) was detected. The aim of this study was to analyze the genetic diversity of Polish E11 environmental and clinical strains circulating between 2017 and 2023 as well as compare them with E11 strains isolated from severe neonatal sepsis cases reported in Europe between 2022 and 2023. Additionally, the study explores the effectiveness of environmental monitoring in tracking the spread of new variants. For this purpose, the complete sequences of the VP1 capsid protein gene were determined for 266 E11 strains isolated in Poland from 2017 to 2023, and phylogenetic analysis was performed. In the years 2017-2023, a significant increase in the detection of E11 strains was observed in both environmental and clinical samples in Poland. The Polish E11 strains represented three different genotypes, C3, D5 and E, and were characterized by a high diversity. In Poland, the intensive circulation of the new variant E11, responsible for severe neonatal infections with a high mortality in Europe, was detected in the years 2022-2023. This investigation demonstrates the important role of environmental surveillance in the tracking of enteroviruses circulation, especially in settings with limited clinical surveillance.
Subject(s)
COVID-19 , Enterovirus B, Human , Phylogeny , SARS-CoV-2 , Poland/epidemiology , Humans , Enterovirus B, Human/genetics , Enterovirus B, Human/classification , Enterovirus B, Human/isolation & purification , COVID-19/epidemiology , COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , Genotype , Genetic Variation , Capsid Proteins/genetics , Infant, Newborn , Echovirus Infections/epidemiology , Echovirus Infections/virology , PandemicsABSTRACT
A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza.
Subject(s)
Antigen Presentation , Capsid Proteins , Hepatitis E virus , Nicotiana , Recombinant Fusion Proteins , Viral Matrix Proteins , Hepatitis E virus/immunology , Hepatitis E virus/genetics , Nicotiana/virology , Nicotiana/genetics , Capsid Proteins/genetics , Capsid Proteins/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Vaccines, Virus-Like Particle/immunology , Vaccines, Virus-Like Particle/genetics , Plants, Genetically Modified , Influenza A virus/immunology , Influenza A virus/genetics , Hepatitis E/immunology , Hepatitis E/prevention & control , Hepatitis E/virology , Viroporin ProteinsABSTRACT
Spiroplasma virus 4 (SpV4) is a bacteriophage of the Microviridae, which packages circular ssDNA within non-enveloped T = 1 icosahedral capsids. It infects spiroplasmas, which are known pathogens of honeybees. Here, the structure of the SpV4 virion is determined using cryo-electron microscopy to a resolution of 2.5 Å. A striking feature of the SpV4 capsid is the mushroom-like protrusions at the 3-fold axes, which is common among all members of the subfamily Gokushovirinae. While the function of the protrusion is currently unknown, this feature varies widely in this subfamily and is therefore possibly an adaptation for host recognition. Furthermore, on the interior of the SpV4 capsid, the location of DNA-binding protein VP8 was identified and shown to have low structural conservation to the capsids of other viruses in the family. The structural characterization of SpV4 will aid future studies analyzing the virus-host interaction, to understand disease mechanisms at a molecular level. Furthermore, the structural comparisons in this study, including a low-resolution structure of the chlamydia phage 2, provide an overview of the structural repertoire of the viruses in this family that infect various bacterial hosts, which in turn infect a wide range of animals and plants.