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Scientific Reports May 2024Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts....
Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus-host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.
Topics: Animals; Metagenomics; Bacteriophages; Gills; Bivalvia; Gene Expression Profiling; Transcriptome; Virome; Bacteria; Symbiosis; Metagenome
PubMed: 38719945
DOI: 10.1038/s41598-024-61272-3 -
Viruses Mar 2024Rabbit hemorrhagic disease virus 2 (RHDV2) emerged in the United States in 2018 and has spread in both domestic and wild rabbits nationwide. The virus has a high...
Rabbit hemorrhagic disease virus 2 (RHDV2) emerged in the United States in 2018 and has spread in both domestic and wild rabbits nationwide. The virus has a high mortality rate and can spread rapidly once introduced in a rabbit population. Vaccination against RHDV2 provides the best protection against disease and should be considered by all rabbit owners. Here, we investigate the duration of immunity provided by vaccination with the Medgene Platform conditionally licensed commercial vaccine 6 months following the initial series. Rabbits received either the vaccination or a placebo and were challenged with RHDV2 6 months later. All vaccinated rabbits survived challenge whereas 18/19 non-vaccinated controls succumbed to infection within 10 or fewer days post-challenge. These results demonstrate lasting immunity following vaccination with the Medgene RHDV2 vaccine.
Topics: Animals; Hemorrhagic Disease Virus, Rabbit; Rabbits; Caliciviridae Infections; Viral Vaccines; Vaccines, Synthetic; Vaccination; Baculoviridae; Antibodies, Viral
PubMed: 38675881
DOI: 10.3390/v16040538 -
Viruses Mar 2024Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly... (Review)
Review
Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.
Topics: Baculoviridae; Recombinant Proteins; Animals; Gene Expression; Bluetongue virus; Genetic Vectors; Virus Assembly; Viral Proteins; Capsid Proteins
PubMed: 38675835
DOI: 10.3390/v16040492 -
Virology Journal Apr 2024Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses....
Stability and integrity of self-assembled bovine parvovirus virus‑like particles (BPV‑VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment.
BACKGROUND
Bovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences.
METHODS
In silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism.
RESULTS
Our findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs.
CONCLUSIONS
In summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.
Topics: Animals; Bocavirus; Baculoviridae; Recombinant Proteins; Vaccines; Parvovirus; Capsid Proteins
PubMed: 38641833
DOI: 10.1186/s12985-024-02322-0 -
Open Veterinary Journal Jan 2024Equine influenza (EI) is a transmissible viral respiratory sickness of the family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for...
BACKGROUND
Equine influenza (EI) is a transmissible viral respiratory sickness of the family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains.
AIM
The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2.
METHODS
In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system.
RESULTS
The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays.
CONCLUSION
In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.
Topics: Horses; Animals; Hemagglutinins; Influenza A Virus, H3N8 Subtype; Influenza A Virus, H7N7 Subtype; Orthomyxoviridae Infections; Vaccines; Baculoviridae
PubMed: 38633177
DOI: 10.5455/OVJ.2024.v14.i1.32 -
Frontiers in Immunology 2024Signal peptide peptidase () is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the...
INTRODUCTION
Signal peptide peptidase () is an intramembrane protease involved in a variety of biological processes, it participates in the processing of signal peptides after the release of the nascent protein to regulate the endoplasmic reticulum associated degradation (ERAD) pathway, binds misfolded membrane proteins, and aids in their clearance process. Additionally, it regulates normal immune surveillance and assists in the processing of viral proteins. Although is essential for many viral infections, its role in silkworms remains unclear. Studying its role in the silkworm, , may be helpful in breeding virus-resistant silkworms.
METHODS
First, we performed RT-qPCR to analyze the expression pattern of . Subsequently, we inhibited using the inhibitor 1,3-di-(N-carboxybenzoyl-L-leucyl-L-leucylaminopropanone ((Z-LL)-ketone) and downregulated the expression of using CRISPR/Cas9 gene editing. Furthermore, we assessed the impact of these interventions on the proliferation of nucleopolyhedrovirus (BmNPV).
RESULTS
We observed a decreased in the expression of during viral proliferation. It was found that higher concentration of the inhibitor resulted in greater inhibition of BmNPV proliferation. The down-regulation of in both in vivo and in vitro was found to affect the proliferation of BmNPV. In comparison to wild type silkworm, silkworms exhibited a 12.4% reduction in mortality rate.
DISCUSSION
Collectively, this work demonstrates that plays a negative regulatory role in silkworm resistance to BmNPV infection and is involved in virus proliferation and replication processes. This finding suggests that servers as a target gene for BmNPV virus resistance in silkworms and can be utilized in resistance breeding programs.
Topics: Animals; Bombyx; Nucleopolyhedroviruses; Gene Editing; Down-Regulation
PubMed: 38585268
DOI: 10.3389/fimmu.2024.1377270 -
Viruses Mar 2024(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch...
(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.
Topics: Animals; Humans; Butyric Acid; HEK293 Cells; Genetic Vectors; Baculoviridae; Plasmids; Mammals
PubMed: 38543791
DOI: 10.3390/v16030426 -
Frontiers in Cellular and Infection... 2024Chagas' is a neglected disease caused by the eukaryotic kinetoplastid parasite, Currently, approximately 8 million people are infected worldwide, most of whom are in...
Chagas' is a neglected disease caused by the eukaryotic kinetoplastid parasite, Currently, approximately 8 million people are infected worldwide, most of whom are in the chronic phase of the disease, which involves cardiac, digestive, or neurologic manifestations. There is an urgent need for a vaccine because treatments are only effective in the initial phase of infection, which is generally underdiagnosed. The selection and combination of antigens, adjuvants, and delivery platforms for vaccine formulations should be designed to trigger mixed humoral and cellular immune responses, considering that has a complex life cycle with both intracellular and bloodstream circulating parasite stages in vertebrate hosts. Here, we report the effectiveness of vaccination with a -specific protein family (TcTASV), employing both recombinant proteins with aluminum hydroxide and a recombinant baculovirus displaying a TcTASV antigen at the capsid. Vaccination stimulated immunological responses by producing lytic antibodies and antigen-specific CD4+ and CD8+ IFNɣ secreting lymphocytes. More than 90% of vaccinated animals survived after lethal challenges with , whereas all control mice died before 30 days post-infection. Vaccination also induced a strong decrease in chronic tissue parasitism and generated immunological memory that allowed vaccinated and infected animals to control both the reactivation of the infection after immunosuppression and a second challenge with . Interestingly, inoculation with baculovirus partially protected the mice against . In brief, we demonstrated for the first time that the combination of the baculovirus platform and the TcTASV family provides effective protection against i, which is a promising vaccine for Chagas disease.
Topics: Humans; Animals; Mice; Parasites; Baculoviridae; Antigens, Protozoan; Chagas Disease; Trypanosoma cruzi; Vaccination; Vaccines; Protozoan Vaccines
PubMed: 38481660
DOI: 10.3389/fcimb.2024.1297321 -
International Journal of Molecular... Feb 2024Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However,...
A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.
Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.
Topics: Animals; Capsid Proteins; Dependovirus; Chromatography, Liquid; Liquid Chromatography-Mass Spectrometry; Workflow; Genetic Vectors; Tandem Mass Spectrometry; Baculoviridae; Mammals
PubMed: 38474031
DOI: 10.3390/ijms25052785 -
Nucleic Acids Research Apr 2024CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components...
CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.
Topics: Animals; Humans; Baculoviridae; CRISPR-Associated Protein 9; CRISPR-Cas Systems; DNA; Gene Editing; Viral Proteins; Cell Line
PubMed: 38412306
DOI: 10.1093/nar/gkae142