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Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models

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A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Invertebrate Viruses".

Deadline for manuscript submissions: closed (15 June 2023) | Viewed by 25573

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Prof. Dr. Miguel López-Ferber

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Hydrosciences Montpellier, Université de Montpellier, IMT Mines Ales, CNRS, IRD, Ales, France
Interests: baculovirus genetic diversity; virus-host interactions; biological control with viruses
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Prof. Dr. Linda King

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Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK
Interests: insect virus biology; baculovirus expression systems; baculovirus-host cell interactions at cellular level; molecular virology; insect virology; the biology and replication of insect baculoviruses in cultured insect cells and in larvae; the role of non-essential genes encoding proteins; the trafficking of virus proteins and particles through insect cells; baculoviruses as gene expression vectors
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Studies on baculoviruses represent a large share of the insect virus research portfolio, partly due to the widespread use of this virus family in biotechnological applications since the landmark studies of Miller and Summers in the 1980s. In recent years, growing knowledge of the replication cycle of these viruses has allowed expanding their use and improving the expression of heterologous genes, both in terms of quantity and quality. The use of baculoviruses for the control of insect pests has been specifically addressed in two recent special issues, but new approaches in this field continue to emerge. Baculoviruses are also used as models for other pathogens, as they possess a narrow host range. In this special issue, we would like to produce a collection of papers showing the variety of applications of this virus family across agriculture, biotechnology, and medicine. We also hope this collection will promote further developments due to the cross-fertilisation of the ideas presented across the various contributions.

Prof. Dr. Miguel López-Ferber
Prof. Dr. Linda King
Guest Editors

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Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, and Virus Genetics Models in Viruses

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16 pages, 6978 KiB

Open AccessArticle

Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells

by Maria Toth, Manuel Reithofer, Gregory Dutra, Patricia Pereira Aguilar, Astrid Dürauer and Reingard Grabherr

Viruses 2024, 16(3), 426; https://doi.org/10.3390/v16030426 - 10 Mar 2024

Viewed by 1639

Abstract

(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. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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13 pages, 2881 KiB

Open AccessArticle

Insecticidal Traits of Variants in a Genotypically Diverse Natural Isolate of Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV)

by Ana Parras-Jurado, Delia Muñoz, Inés Beperet, Trevor Williams and Primitivo Caballero

Viruses 2023, 15(7), 1526; https://doi.org/10.3390/v15071526 - 10 Jul 2023

Cited by 1 | Viewed by 1519

Abstract

Outbreaks of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), a major pest of soybean, can be controlled below economic thresholds with methods that do not involve the application of synthetic insecticides. Formulations based on natural isolates of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (Baculoviridae: Alphabaculovirus) played a significant role in integrated pest management programs in the early 2000s, but a new generation of chemical insecticides and transgenic soybean have displaced AgMNPV-based products over the past decade. However, the marked genotypic variability present among and within alphabaculovirus isolates suggests that highly insecticidal genotypic variants can be isolated and used to reduce virus production costs or overcome isolate-dependent host resistance. This study aimed to select novel variants of AgMNPV with suitable insecticidal traits that could complement the existing AgMNPV active ingredients. Three distinct AgMNPV isolates were compared using their restriction endonuclease profile and in terms of their occlusion body (OB) pathogenicity. One isolate was selected (AgABB51) from which eighteen genotypic variants were plaque purified and characterized in terms of their insecticidal properties. The five most pathogenic variants varied in OB pathogenicity, although none of them was faster-killing or had higher OB production characteristics than the wild-type isolate. We conclude that the AgABB51 wild-type isolates appear to be genotypically structured for fast speed of kill and high OB production, both of which would favor horizontal transmission. Interactions among the component variants are likely to influence this insecticidal phenotype. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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15 pages, 1798 KiB

Open AccessArticle

Evaluating Novel Quantification Methods for Infectious Baculoviruses

by Keven Lothert, Elena Bagrin and Michael W. Wolff

Viruses 2023, 15(4), 998; https://doi.org/10.3390/v15040998 - 19 Apr 2023

Cited by 1 | Viewed by 2971

Abstract

Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring. Accordingly, flow cytometry and quantitative polymerase chain reaction have attracted increasing interest in recent years, offering various advantages for rapid quantification. Here, we compared different approaches for the assessment of infectious viruses, using a model baculovirus. Firstly, infectivity was estimated by the quantification of viral nucleic acids in infected cells, and secondly, different flow cytometric approaches were investigated regarding analysis times and calibration ranges. The flow cytometry technique included a quantification based on post-infection fluorophore expression and labeling of a viral surface protein using fluorescent antibodies. Additionally, the possibility of viral (m)RNA labeling in infected cells was investigated as a proof of concept. The results confirmed that infectivity assessment based on qPCR is not trivial and requires sophisticated method optimization, whereas staining of viral surface proteins is a fast and feasible approach for enveloped viruses. Finally, labeling of viral (m)RNA in infected cells appears to be a promising opportunity but will require further research. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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20 pages, 3321 KiB

Open AccessArticle

Transcriptional Reprogramming of Autographa Californica Multiple Nucleopolyhedrovirus Chitinase and Cathepsin Genes Enhances Virulence

by Jeffrey J. Hodgson, A. Lorena Passarelli and Peter J. Krell

Viruses 2023, 15(2), 503; https://doi.org/10.3390/v15020503 - 11 Feb 2023

Cited by 2 | Viewed by 1702

Abstract

The baculoviral chitinase (CHIA) and cathepsin (V-CATH) enzymes promote terminal insect host liquefaction, which aids viral progeny dissemination. Recombinant Autographa californica nucleopolyhedrovirus (AcMNPV)-derived viruses were previously generated with reprogrammed chiA transcription by replacing the native promoter with the AcMNPV polyhedrin (polh) or core protein (p6.9) promoter sequences, but of both these chiA-reprogrammed viruses lacked v-cath transcription and V-CATH enzymatic activity. Here, we report that dual p6.9/polh promoter reprogramming of the adjacent chiA/v-cath genes resulted in modulated temporal transcription of both genes without impacting infectious budded virus production. These promoter changes increased CHIA and V-CATH enzyme activities in infected Spodoptera frugiperda-derived cultured cells and Trichoplusia ni larvae. In addition, larvae infected with the dual reprogrammed virus had earlier mortalities and liquefaction. This recombinant baculovirus, lacking exogenous genomic elements and increased chiA/v-cath expression levels, may be desirable for and amenable to producing enhanced baculovirus-based biopesticides. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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15 pages, 4558 KiB

Open AccessArticle

Integrated Analysis of MicroRNA and mRNA Expression Profiles in the Fat Bodies of MbMNPV-Infected Helicoverpa armigera

by Zhenpu Liang, Yanqing Yang, Xiaoyan Sun, Junyang Du, Qiuyun Wang, Guozhi Zhang, Jiran Zhang, Xinming Yin, Deepali Singh, Ping Su and Xiaoxia Zhang

Viruses 2023, 15(1), 19; https://doi.org/10.3390/v15010019 - 21 Dec 2022

Cited by 5 | Viewed by 1711

Abstract

MicroRNAs (miRNAs), are a novel class of gene expression regulators, that have been found to participate in regulating host–virus interactions. However, the function of insect-derived miRNAs in response to virus infection is poorly understood. We analyzed miRNA expression profiles in the fat bodies of Helicoverpa armigera (H. armigera) infected with Mamestra brassicae multiple nucleopolyhedroviruses (MbMNPV). A total of 52 differentially expressed miRNAs (DEmiRNAs) were filtered out through RNA-seq analysis. The targets of 52 DEmiRNAs were predicted and 100 miRNA–mRNA interaction pairs were obtained. The predicted targets of DEmiRNAs were mainly enriched in the Wnt signaling pathway, phagosome, and mTOR signaling pathway, which are related to the virus infection. Real-time PCR was used to verify the RNA sequencing results. ame-miR-317-3p, mse-miR-34, novel1-star, and sfr-miR-6094-5p were shown to be involved in the host response to MbMNPV infection. Results suggest that sfr-miR-6094-5p can negatively regulate the expression of four host genes eIF3-S7, CG7583, CG16901, and btf314, and inhibited MbMNPV infection significantly. Further studies showed that RNAi-mediated knockdown of eIF3-S7 inhibited the MbMNPV infection. These findings suggest that sfr-miR-6094-5p inhibits MbMNPV infection by negatively regulating the expression of eIF3-S7. This study provides new insights into MbMNPV and H. armigera interaction mechanisms. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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14 pages, 3113 KiB

Open AccessArticle

Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System

by Joaquín Poodts, Ignacio Smith, Joaquín Manuel Birenbaum, María Sol Rodriguez, Luciano Montero, Federico Javier Wolman, Juan Ignacio Marfía, Silvina Noemí Valdez, Leonardo Gabriel Alonso, Alexandra Marisa Targovnik and María Victoria Miranda

Viruses 2022, 14(12), 2794; https://doi.org/10.3390/v14122794 - 15 Dec 2022

Cited by 4 | Viewed by 2521

Abstract

Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host’s cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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13 pages, 4360 KiB

Open AccessArticle

Successful Rescue of Synthetic AcMNPV with a ~17 kb Deletion in the C1 Region of the Genome

by Yijia Guo, Hengrui Hu, Han Xiao, Fei Deng, Jiang Li, Manli Wang and Zhihong Hu

Viruses 2022, 14(12), 2780; https://doi.org/10.3390/v14122780 - 13 Dec 2022

Cited by 1 | Viewed by 2066

Abstract

Baculoviruses have been widely used as expression vectors. However, numerous genes in the baculoviral genome are non-essential for cellular infection and protein expression, making the optimisation of baculovirus expression vectors possible. We used a synthetic biological method to reduce the number of genes in a partial region of the autograph californica multiple nucleopolyhedrovirus (AcMNPV), the most widely used baculovirus expression vector. The C1 region of the AcMNPV is 46.4 kb and is subdivided into B1, B2, and B3 fragments. We first designed modified B1, B2, and B3 fragments by deleting the non-essential genes, and then synthesised complete viral genomes containing either individual modified B fragments or joint modified B fragments through transformation-related recombination in yeast. The synthetic genomes were then transfected into Sf9 cells to rescue the progeny viruses and test their infectivity. The design-build-test cycle was repeated until the ultimately rescued virus could produce progeny viruses efficiently. Finally, AcMNPV-Syn-mC1-1.1 by deleting approximately 17.2 kb, including 20 ORFs, in the C1 region, was obtained. This is essential to the synthesis of a minimal AcMNPV genome that can generate infectious progeny viruses and can be further used to optimise the foundation of baculovirus expression vectors. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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18 pages, 3086 KiB

Open AccessArticle

Utility of Alternative Promoters for Foreign Gene Expression Using the Baculovirus Expression Vector System

by Mark R. Bruder and Marc G. Aucoin

Viruses 2022, 14(12), 2670; https://doi.org/10.3390/v14122670 - 29 Nov 2022

Cited by 6 | Viewed by 2403

Abstract

The baculovirus expression vector system (BEVS) is a widely used platform for recombinant protein production for use in a wide variety of applications. Of particular interest is production of virus-like particles (VLPs), which consist of multiple viral proteins that self-assemble in strict stoichiometric ratios to mimic the structure of a virus but lacks its genetic material, while a significant amount of effort has been spent on optimizing expression ratios by co-infecting cells with multiple recombinant BEVs and modulating different process parameters, co-expressing multiple foreign genes from a single rBEV may offer more promise. However, there is currently a lack of promoters available with which to optimize co-expression of each foreign gene. To address this, previously published transcriptome data was used to identify promoters that have incrementally lower expression profiles and compared by expressing model cytoplasmic and secreted proteins. Bioinformatics was also used to identify sequence determinants that may be important for late gene transcription regulation, and translation initiation. The identified promoters and bioinformatics analyses may be useful for optimizing expression of foreign genes in the BEVS. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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14 pages, 2227 KiB

Open AccessArticle

Overexpression Bombyx mori HEXIM1 Facilitates Immune Escape of Bombyx mori Nucleopolyhedrovirus by Suppressing BmRelish-Driven Immune Responses

by Guanping Chen, Yuedong Li, Xiangshuo Kong, Shudi Zhao, Jiale Li and Xiaofeng Wu

Viruses 2022, 14(12), 2636; https://doi.org/10.3390/v14122636 - 25 Nov 2022

Viewed by 1770

Abstract

Bombyx mori nucleopolyhedrovirus (BmNPV), a typical arthropod-specific enveloped DNA virus, is one of the most serious pathogens in silkworm farming, but the potential mechanisms of the evasion of innate immune responses from BmNPV infection are still poorly understood. HEXIM1 is an RNA-binding protein, best known as an inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation by RNA polymerase II. In this study, Bombyx mori HEXIM1 (BmHEXIM1) was cloned and characterized, and its expression was found to be remarkably upregulated after BmNPV infection. Furthermore, BmHEXIM1 was detected to increase the proliferation of BmNPV, and its full length is essential for assisting BmNPV immune escape by suppressing BmRelish-driven immune responses. This study brought new insights into the mechanisms of immune escape of BmNPV and provided theoretical guidance for the breeding of BmNPV-resistant silkworm varieties. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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19 pages, 953 KiB

Open AccessReview

The History of Baculovirology in Africa

by Sean Moore and Michael Jukes

Viruses 2023, 15(7), 1519; https://doi.org/10.3390/v15071519 - 7 Jul 2023

Cited by 2 | Viewed by 1658

Abstract

Baculovirology has been studied on the African continent for the development of insect virus-based biopesticides and, to a much lesser extent, vaccine production and delivery, since the 1960s. In this review, we focus only on baculoviruses as biopesticides for agricultural pests in Africa. At least 11 species of baculovirus have been discovered or studied on the African continent, some with several distinct isolates, with the objective in most cases being the development of a biopesticide. These include the nucleopolyhedroviruses of Helicoverpa armigera, Cryptophlebia peltastica, Spodoptera exempta, Spodoptera frugiperda, Spodoptera littoralis, and Maruca vitrata, as well as the granuloviruses of Cydia pomonella, Plutella xylostella, Thaumatotibia (Cryptophlebia) leucotreta, Choristoneura occidentalis, and Phthorimaea operculella. Eleven different baculovirus-based biopesticides are recorded as being registered and commercially available on the African continent. Baculoviruses are recorded to have been isolated, researched, utilised in field trials, and/or commercially deployed as biopesticides in at least 13 different African countries. Baculovirus research is ongoing in Africa, and researchers are confident that further novel species and isolates will be discovered, to the benefit of environmentally responsible agricultural pest management, not only in Africa but also elsewhere. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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17 pages, 848 KiB

Open AccessReview

Advances in CRISPR-Cas9 for the Baculovirus Vector System: A Systematic Review

by Duygu Sari-Ak, Omar Alomari, Raghad Al Shomali, Jackwee Lim and Deepak B. Thimiri Govinda Raj

Viruses 2023, 15(1), 54; https://doi.org/10.3390/v15010054 - 24 Dec 2022

Cited by 1 | Viewed by 4625

Abstract

The baculovirus expression vector systems (BEVS) have been widely used for the recombinant production of proteins in insect cells and with high insert capacity. However, baculovirus does not replicate in mammalian cells; thus, the BacMam system, a heterogenous expression system that can infect certain mammalian cells, was developed. Since then, the BacMam system has enabled transgene expression via mammalian-specific promoters in human cells, and later, the MultiBacMam system enabled multi-protein expression in mammalian cells. In this review, we will cover the continual development of the BEVS in combination with CRPISPR-Cas technologies to drive genome-editing in mammalian cells. Additionally, we highlight the use of CRISPR-Cas in glycoengineering to potentially produce a new class of glycoprotein medicines in insect cells. Moreover, we anticipate CRISPR-Cas9 to play a crucial role in the development of protein expression systems, gene therapy, and advancing genome engineering applications in the future. Full article

(This article belongs to the Special Issue Applications of Baculoviruses: Expression Factories, Vaccines and VLPs, Gene Delivery Vectors, Biological Control and Virus Genetics Models)

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