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1.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474031

RESUMO

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 cap 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.


Assuntos
Proteínas do Capsídeo , Dependovirus , Animais , Proteínas do Capsídeo/genética , Dependovirus/genética , Cromatografia Líquida , Fluxo de Trabalho , Vetores Genéticos , Espectrometria de Massas em Tandem , Baculoviridae/genética , Mamíferos/metabolismo
2.
Front Cell Infect Microbiol ; 14: 1297321, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38481660

RESUMO

Chagas' is a neglected disease caused by the eukaryotic kinetoplastid parasite, Trypanosoma cruzi. 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 T. cruzi 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 T. cruzi-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+ IFNÉ£ secreting lymphocytes. More than 90% of vaccinated animals survived after lethal challenges with T. cruzi, 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 T. cruzi. Interestingly, inoculation with wild-type baculovirus partially protected the mice against T. cruzi. In brief, we demonstrated for the first time that the combination of the baculovirus platform and the TcTASV family provides effective protection against Trypanosoma cruzi, which is a promising vaccine for Chagas disease.


Assuntos
Doença de Chagas , Parasitos , Vacinas Protozoárias , Trypanosoma cruzi , Vacinas , Humanos , Animais , Camundongos , Baculoviridae/genética , Antígenos de Protozoários/genética , Doença de Chagas/parasitologia , Trypanosoma cruzi/genética , Vacinação , Vacinas Protozoárias/genética
3.
J Gen Virol ; 105(2)2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38314674

RESUMO

Baculoviruses are highly host specific, and their host range is usually restricted to a single or a few closely related insect species, except for few virus species, e.g. Alphabaculovirus aucalifonicae and Alphabaculovirus mabrassicae. In this study, two new alphabaculovirus isolates were isolated from the larvae of Mamestra brassicae and Mythimna separata, which were named as Mamestra brassicae multiple nucleopolyhedrovirus isolate QD (MbMNPV-QD) and Mythimna separata multiple nucleopolyhedrovirus isolate Hb (MyseMNPV-Hb), respectively. The Kimura two-parameter values based on the concatenated 38 core genes of baculovirus revealed that MbMNPV (isolates QD/CHb1/K1/CTa), MyseMNPV-Hb, Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) and Mamestra configurata nucleopolyhedrovirus B (MacoNPV-B) were different isolates of a same virus species. A phylogenetic tree of baculoviruses and nudiviruses constructed from their 20 homologous gene sequences, and that of their isolated hosts constructed from 13 protein-coding genes of the insect mitochondrial genomes, were used to analyse the coevolution of baculoviruses with their isolated hosts. The results showed that M. brassicae was the most likely ancestral host of these virus isolates, included MbMNPV isolates, MyseMNPV-Hb, HearMNPV, and MacoNPV-B. Therefore, we concluded that these virus isolates belong to the existing virus species - Alphabaculovirus mabrassicae with M. brassicae as their ancestral host.


Assuntos
Mariposas , Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/genética , Filogenia , Larva , Baculoviridae , Especificidade de Hospedeiro , Insetos
4.
Open Biol ; 14(2): 230278, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38378139

RESUMO

Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Spodoptera exigua caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar's central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.


Assuntos
Baculoviridae , Sistema Nervoso Central , Nucleopoliedrovírus , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Spodoptera/metabolismo , Sistema Nervoso Central/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo
5.
Viruses ; 16(2)2024 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-38400028

RESUMO

Baculoviridae, a virus family characterized by a single large double stranded DNA, encompasses the majority of viral bioinsecticides, representing a highly promising and environmentally friendly pesticide approach to insect control. This study focuses on the characterization of a baculovirus isolated from larvae of Calliteara abietis (Erebidae, Lymantriidae) collected in Mongolian pinaceae forests. This new isolate was called Calliteara abietis nucleopolyhedrovirus (CaabNPV). CaabNPV exhibits an irregular polyhedron shape, and significant variation in the diameter of its occlusion bodies (OBs) was observed. Nucleotide distance calculations confirmed CaabNPV as a novel baculovirus. The CaabNPV genome spans 177,161 bp with a G+C content of 45.12% and harbors 150 potential open reading frames (ORFs), including 38 core genes. A comprehensive genomic analysis categorizes CaabNPV within Group II alphabaculovirus, revealing a close phylogenetic relationship with Alphabaculovirus orleucostigmae (OrleNPV). Additionally, repeat sequence analysis identified three highly repetitive sequences consisting of 112 bp repeat units, known as homologous regions (hrs). This research contributes valuable insights into CaabNPV's phylogenetic placement, genomic structure, and its potential applications in insect biocontrol.


Assuntos
Mariposas , Nucleopoliedrovírus , Animais , Baculoviridae/genética , Filogenia , Genoma Viral , Análise de Sequência de DNA , Fases de Leitura Aberta
6.
Methods Mol Biol ; 2772: 311-322, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38411825

RESUMO

The overproduction of proteins of the endoplasmic reticulum (ER) of plant cells in prokaryotic heterologous gene expression system remains a technical challenge. Recent advances in genetically modified insect cell technology and virus engineering methods have paved the way to produce recombinant ER plant proteins, including those harboring posttranslational modifications, and therefore, to yield ER plant proteins that are natively folded and fully functional. The present contribution focuses on the baculovirus-expression system flashBAC, which overcomes certain technical hurdles found in other insect cell-based expression systems such as the generation of a bacmid and the negative selection of recombinant clones.


Assuntos
Arabidopsis , Arabidopsis/genética , Baculoviridae/genética , Proteínas de Plantas , Células Clonais , Retículo Endoplasmático
7.
J Gen Virol ; 105(2)2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38376497

RESUMO

Baculoviruses are insect-specific pathogens. Novel baculovirus isolates provide new options for the biological control of pests. Therefore, research into the biological characteristics of newly isolated baculoviruses, including accurate classification and nomenclature, is important. In this study, a baculovirus was isolated from Mythimna separata and its complete genome sequence was determined by next-generation sequencing. The double-stranded DNA genome was 153 882 bp in length, encoding 163 open reading frames. The virus was identified as a variant of Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) and designated Mamestra brassicae multiple nucleopolyhedrovirus CHN1 (MbMNPV-CHN1) according to ultrastructural analysis, genome comparison and phylogenetic analysis. Phylogenetic inference placed MbMNPV-CHN1 in a clade containing isolates of MacoNPV-A, MacoNPV-B and MbMNPV, which we have designated the Mb-McNPV group. The genomes of isolates in the Mb-McNPV group exhibited a high degree of collinearity with relatively minor differences in the content of annotated open reading frames. The development of codon usage bias in the Mb-McNPV group was affected mainly by natural selection. MbMNPV-CHN1 shows high infectivity against seven species of Lepidoptera. The yield of MbMNPV-CHN1 in the fourth- and fifth-instar M. separata larvae was 6.25×109-1.23×1010 OBs/cadaver. Our data provide insights into the classification, host range and virulence differences among baculoviruses of the Mb-McNPV group, as well as a promising potential new baculoviral insecticide.


Assuntos
Doença de Charcot-Marie-Tooth , Lepidópteros , Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/genética , Filogenia , Baculoviridae/genética , Evolução Biológica
8.
Sheng Wu Gong Cheng Xue Bao ; 40(2): 473-484, 2024 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-38369834

RESUMO

Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key bottlenecks such as low yield and high-cost. The aim of this study was to establish a technology system for production of AAV in the double virus infected insects by using multiple-gene deleted baculovirus. First, a multiple gene deleted baculovirus for AAV production was constructed, and the baculovirus titer and its effect on infected cells was examined. Subsequently, the insect cells were co-infected with the double baculovirus and the infection conditions were optimized. At the final stage, we performed AAV production based on optimized conditions, and evaluated relevant parameters including production titer and quality. The results showed that the titer of AAV produced in the multiple gene deleted baculovirus was not different from that of the wild type, but the rate of cell death was significantly slower upon infection. Using the double virus route for optimized production of AAV, the genome titers were 1.63×1011 VG/mL for Bac4.0-1 and 1.02×1011 VG/mL for Bac5.0-2, which were elevated 240% and 110%, respectively, compared with that of the wild-type. Electron microscopy observations revealed that all three groups exhibited normal AAV viral morphology and they showed similar transduction activity. Taken together, we developed an AAV production system based on the infection of insect cells using multiple-gene deleted baculovirus, which significantly improved the virus yield and showed application potential.


Assuntos
Baculoviridae , Dependovirus , Animais , Dependovirus/genética , Dependovirus/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Linhagem Celular , Vetores Genéticos , Insetos/genética
9.
N Biotechnol ; 80: 46-55, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38302001

RESUMO

The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.


Assuntos
Baculoviridae , Baculoviridae/metabolismo , Linhagem Celular , Proliferação de Células , Contagem de Células
10.
Methods Mol Biol ; 2762: 43-70, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38315359

RESUMO

The baculovirus/insect cell expression system is a very useful tool for reagent and antigen generation in vaccinology, virology, and immunology. It allows for the production of recombinant glycoproteins, which are used as antigens in vaccination studies and as reagents in immunological assays. Here, we describe the process of recombinant glycoprotein production using the baculovirus/insect cell expression system.


Assuntos
Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Proteínas Recombinantes , Insetos/metabolismo
11.
PLoS One ; 19(1): e0285651, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38180986

RESUMO

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Cálcio , Humanos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Baculoviridae/genética , Biofísica , Sinalização do Cálcio , Escherichia coli/genética
12.
J Invertebr Pathol ; 203: 108062, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38244836

RESUMO

In vitro propagation of shrimp viruses IHHNV (Infectious hematopoietic hypodermal necrosis virus)/PstDV1 and MBV (Monodon baculovirus)/PmNV in the primary haemocyte culture was successfully be accomplished in the present study. The haemocytes were maintained in double-strength L15 medium with growth supplements and antibiotics. Cytopathic changes observed in the cells were detachment, slendering of cells, vacuolation, and rounding of the cells. Replication of viruses in the culture was confirmed through the expression of DNA polymerase, polyhedrin and lef 4 of MBV and capsid gene of IHHNV. Ultrathin sections of haemocytes observed through electron microscopy revealed scattered chromatin, virogenic stroma and presence of viruses and occlusion bodies (MBV). The study revealed that primary haemocyte culture could be used as a platform for the propagation of a variety of shrimp viruses.


Assuntos
Densovirinae , Penaeidae , Animais , Baculoviridae/genética , Densovirinae/genética , Crustáceos , Necrose
13.
Int J Mol Sci ; 25(1)2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38203774

RESUMO

Baculoviruses are viral pathogens that infect different species of Lepidoptera, Diptera, and Hymenoptera, with a global distribution. Due to their biological characteristics and the biotechnological applications derived from these entities, the Baculoviridae family is an important subject of study and manipulation in the natural sciences. With the advent of RNA interference mechanisms, the presence of baculoviral genes that do not code for proteins but instead generate transcripts similar to microRNAs (miRNAs) has been described. These miRNAs are functionally associated with the regulation of gene expression, both in viral and host sequences. This article provides a comprehensive review of miRNA biogenesis, function, and characterization in general, with a specific focus on those identified in baculoviruses. Furthermore, it delves into the specific roles of baculoviral miRNAs in regulating viral and host genes and presents structural and thermodynamic stability studies that are useful for detecting shared characteristics with predictive utility. This review aims to expand our understanding of the baculoviral miRNAome, contributing to improvements in the production of baculovirus-based biopesticides, management of resistance phenomena in pests, enhancement of recombinant protein production systems, and development of diverse and improved BacMam vectors to meet biomedical demands.


Assuntos
MicroRNAs , MicroRNAs/genética , Baculoviridae/genética , Interferência de RNA , Agentes de Controle Biológico , Biotecnologia
14.
BMC Genomics ; 25(1): 91, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38253995

RESUMO

BACKGROUND: Spodoptera litura is a harmful pest that feeds on more than 80 species of plants, and can be infected and killed by Spodoptera litura nucleopolyhedrovirus (SpltNPV). SpltNPV-C3 is a type C SpltNPV clone, that was observed and collected in Japan. Compared with type A or type B SpltNPVs, SpltNPV-C3 can cause the rapid mortality of S. litura larvae. METHODS: In this study, occlusion bodies (OBs) and occlusion-derived viruses (ODVs) of SpltNPV-C3 were purified, and OBs were observed by scanning electron microscopy (SEM). ODVs were observed under a transmission electron microscope (TEM). RESULTS: Both OBs and ODVs exhibit morphological characteristics typical of nucleopolyhedroviruses (NPVs).The genome of SpltNPV-C3 was sequenced and analyzed; the total length was 148,634 bp (GenBank accession 780,426,which was submitted as SpltNPV-II), with a G + C content of 45%. A total of 149 predicted ORFs were found. A phylogenetic tree of 90 baculoviruses was constructed based on core baculovirus genes. LC‒MS/MS was used to analyze the proteins of SpltNPV-C3; 34 proteins were found in the purified ODVs, 15 of which were core proteins. The structure of the complexes formed by per os infectivity factors 1, 2, 3 and 4 (PIF-1, PIF-2, PIF-3 and PIF-4) was predicted with the help of the AlphaFold multimer tool and predicted conserved sequences in PIF-3. SpltNPV-C3 is a valuable species because of its virulence, and the analysis of its genome and proteins in this research will be beneficial for pest control efforts.


Assuntos
Nucleopoliedrovírus , Proteoma , Animais , Nucleopoliedrovírus/genética , Spodoptera , Cromatografia Líquida , Filogenia , Espectrometria de Massas em Tandem , Baculoviridae
15.
Nat Commun ; 15(1): 250, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38177118

RESUMO

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 Å helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.


Assuntos
Baculoviridae , Nucleocapsídeo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Spodoptera , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo
16.
Gene Ther ; 31(3-4): 187-194, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38278988

RESUMO

Baculoviruses have shown great potential as gene delivery vectors in mammals, although their effectiveness in transferring genes varies across different cell lines. A widely employed strategy to improve transduction efficiency is the pseudotyping of viral vectors. In this study, we aimed to develop a stable Sf9 insect cell line that inducibly expresses the G-protein of the vesicular stomatitis virus to pseudotype budded baculoviruses. It was obtained by inserting the VSV-G gene under the control of the very strong and infection-inducible pXXL promoter and was subsequently diluted to establish oligoclonal lines, which were selected by the fusogenic properties of VSV-G and its expression levels in infected cells and purified budded virions. Next, to enhance the performance of the cell line, the infection conditions under which functional pseudotyped baculoviruses are obtained were optimized. Finally, different baculoviruses were pseudotyped and the expression of the transgene was quantified in mammalian cells of diverse origins using flow cytometry. The transduction efficiency of pseudotyped baculovirus consistently increased across all tested mammalian cell lines compared with control viruses. These findings demonstrate the feasibility and advantages of improving gene delivery performance without the need to insert the pseudotyping gene into the baculoviral genomes.


Assuntos
Baculoviridae , Técnicas de Transferência de Genes , Animais , Baculoviridae/genética , Linhagem Celular , Terapia Genética , Regiões Promotoras Genéticas , Vetores Genéticos/genética , Transdução Genética , Proteínas do Envelope Viral/genética , Mamíferos/genética , Mamíferos/metabolismo
17.
Mol Biotechnol ; 66(2): 354-364, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37162721

RESUMO

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.


Assuntos
Compostos de Amônio , Vírus da Raiva , Raiva , Animais , Células Sf9 , Vírus da Raiva/genética , Glutamina , Baculoviridae/genética , Proteínas Recombinantes/genética , Meios de Cultura Livres de Soro , Ácido Glutâmico , Lactatos , Glucose , Spodoptera
18.
Protein Expr Purif ; 215: 106406, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37995943

RESUMO

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.


Assuntos
Baculoviridae , Triagem , Fluxo de Trabalho , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas Recombinantes , Vetores Genéticos
19.
Front Cell Infect Microbiol ; 13: 1291433, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38076451

RESUMO

The fall armyworm (FAW), Spodoptera frugiperda, has been the most devastating pest of corn as well as of other crops in America, and more recently in Africa and Asia. The development of resistance to chemical insecticides led the search for environmentally friendly biological alternatives such as baculoviruses. This study focuses on the primary infection of the baculovirus SfNPV-Ar in the FAW's midgut epithelium, by analyzing the differential expression of transcripts in excised midguts at 6, 12, and 24 h post-infection (hpi), and predicted their interactions. Interaction of viral factors with the infected midgut tissue could alters various cellular processes, such as the apoptotic system due to the up-regulation observed of FABP at 6 hpi and of HSP90 at 24 hpi, along with the down-regulated PRX at 6 hpi and FABP transcripts between 12 and 24 hpi. Changes in transcript regulation could affect the cellular architecture of infected cells due to up-regulation of ARP 2/3 at 6 and 12 hpi, followed by down-regulation at 24 hpi. In relation to protein folding proteins, HSP90 was up-regulated at 24 hpi and PDI was down-regulated between 6 and 12 hpi. With respect to metabolism and cellular transport, AcilBP and ATPS0 were up regulated at 6 hpi and 12 hpi, respectively. In reference to transcription and translation up-regulation of RPL11 at 6 hpi and of FPN32 and RPL19 at 24 hpi was detected, as well as the down-regulation of RPL19 at 6 hpi, of PDI and RPL7 at 12 hpi, and of FABP at 24 hpi. In conclusion, gene regulation induced by viral infection could be related to the cytoskeleton and cellular metabolism as well as to oxidative stress, apoptosis, protein folding, translation, and ribosomal structure. The results presented in this work are an approach to understanding how the virus takes control of the general metabolism of the insect host during the primary infection period.


Assuntos
Baculoviridae , Inseticidas , Animais , Baculoviridae/genética , Spodoptera/genética , Larva , Perfilação da Expressão Gênica , Inseticidas/farmacologia
20.
Nat Commun ; 14(1): 7481, 2023 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-37980340

RESUMO

Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used as a bioinsecticide and a protein expression vector. Despite their importance, very little is known about the structure of most baculovirus proteins. Here, we show a 3.2 Å resolution structure of helical cylindrical body of the AcMNPV nucleocapsid, composed of VP39, as well as 4.3 Å resolution structures of both the head and the base of the nucleocapsid composed of over 100 protein subunits. AcMNPV VP39 demonstrates some features of the HK97-like fold and utilizes disulfide-bonds and a set of interactions at its C-termini to mediate nucleocapsid assembly and stability. At both ends of the nucleocapsid, the VP39 cylinder is constricted by an outer shell ring composed of proteins AC104, AC142 and AC109. AC101(BV/ODV-C42) and AC144(ODV-EC27) form a C14 symmetric inner layer at both capsid head and base. In the base, these proteins interact with a 7-fold symmetric capsid plug, while a portal-like structure is seen in the central portion of head. Additionally, we propose an application of AlphaFold2 for model building in intermediate resolution density.


Assuntos
Baculoviridae , Nucleocapsídeo , Animais , Baculoviridae/metabolismo , Microscopia Crioeletrônica , Spodoptera , Nucleocapsídeo/metabolismo , Proteínas do Capsídeo/metabolismo
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