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Baculovirus Advances and Applications

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

Deadline for manuscript submissions: closed (30 September 2018) | Viewed by 56827

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Prof. Dr. Linda King

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Guest Editor

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
Special Issues, Collections and Topics in MDPI journals

Prof. Robert Possee

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Guest Editor

Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK
Interests: molecular virology; insect baculoviruses; baculovirus expression vector systems

Special Issue Information

Dear Colleagues,

It is now more than 30 years since the use of baculoviruses as expression vectors was first described. Since then, many hundreds, if not thousands, of genes have been expressed in insect cells using the baculovirus system. Advances have been made to enable multiple genes to be expressed that can assemble into protein complexes including virus-like particles. High yields of quality proteins including those targeted to membranes or for secretion can be attained. The vectors have also been modified to facilitate gene expression in mammalian and human cells. These developments have enabled the insect expression system to be at the forefront of developments to produce new vaccines for human and animal health, and as a vector for the delivery of genes into human tissue. It is these developments that form the focus of this Special Issue.

Prof. Linda King
Prof. Robert Possee
Guest Editors

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Keywords

baculovirus expression vectors
BacMam vectors
insect cells
mammalian cells
virus-like particles
vaccines
gene therapy

Published Papers (10 papers)

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Research

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17 pages, 2949 KiB

Open AccessArticle

Autographa Californica Multiple Nucleopolyhedrovirus Enters Host Cells via Clathrin-Mediated Endocytosis and Direct Fusion with the Plasma Membrane

by Fujun Qin, Congrui Xu, Chengfeng Lei, Jia Hu and Xiulian Sun

Viruses 2018, 10(11), 632; https://doi.org/10.3390/v10110632 - 14 Nov 2018

Cited by 5 | Viewed by 3446

Abstract

The cell entry mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not fully understood. Previous studies showed that AcMNPV entered host cells primarily through clathrin-mediated endocytosis, and could efficiently infect cells via fusion with the plasma membrane after a low-pH trigger. However, whether AcMNPV enters cells via these two pathways simultaneously, and the exact manner in which AcMNPV particles are internalized into cells remains unclear. In this study, using single-virus tracking, we observed that AcMNPV particles were first captured by pre-existing clathrin-coated pits (CCP), and were then delivered to early endosomes. Population-based analysis of single-virus tracking and quantitative electron microscopy demonstrated that the majority of particles were captured by CCPs and internalized via invagination. In contrast, a minority of virus particles were not delivered to CCPs, and were internalized through direct fusion with the plasma membrane without invagination. Quantitative electron microscopy also showed that, while inhibition of CCP assembly significantly impaired viral internalization, inhibition of endosomal acidification blocked virus particles out of vesicles. Collectively, these findings demonstrated that approximately 90% of AcMNPV particles entered cells through clathrin-mediated endocytosis and 10% entered via direct fusion with the plasma membrane. This study will lead toward a better understanding of AcMNPV infection. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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17 pages, 3042 KiB

Open AccessArticle

Improved Baculovirus Vectors for Transduction and Gene Expression in Human Pancreatic Islet Cells

by Leo P. Graves, Mine Aksular, Riyadh A. Alakeely, Daniel Ruiz Buck, Adam C. Chambers, Fernanda Murguia-Meca, Juan-Jose Plata-Muñoz, Stephen Hughes, Paul R. V. Johnson, Robert D. Possee and Linda A. King

Viruses 2018, 10(10), 574; https://doi.org/10.3390/v10100574 - 20 Oct 2018

Cited by 5 | Viewed by 5796

Abstract

Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury, gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here, a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet β cells (EndoC βH3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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14 pages, 5876 KiB

Open AccessArticle

Disruption of Autographa Californica Multiple Nucleopolyhedrovirus ac111 Results in Reduced per os Infectivity in a Host-Dependent Manner

by Sainan Li, Lu Li, Haizhou Zhao and Wenhua Liu

Viruses 2018, 10(10), 527; https://doi.org/10.3390/v10100527 - 27 Sep 2018

Cited by 3 | Viewed by 2696

Abstract

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac111 gene is highly conserved in lepidopteran-specific baculoviruses, and its function in the AcMNPV life cycle is still unknown. To investigate the function of ac111, an ac111-knockout AcMNPV (vAc111KO) was constructed through homologous recombination in Escherichia coli. Viral growth curve analysis and plaque assays showed that the deletion of ac111 had no effect on infectious budded virion production. Quantitative real-time polymerase chain reaction analysis confirmed that viral DNA replication was unaffected in the absence of ac111. Electron microscopy revealed that the ac111 deletion did not affect nucleocapsid assembly, occlusion-derived virion formation, or the embedding of occlusion-derived virions into the occlusion bodies. However, in vivo bioassays showed that although the deletion of ac111 did not affect the per os infectivity of AcMNPV in Spodoptera exigua larvae, it led to an approximately five-fold reduction in infectivity of AcMNPV in Trichoplusia ni larvae, and vAc111KO took approximately 21 h longer to kill Trichoplusia ni larvae than the wild-type viruses. Taken together, our results demonstrated that although ac111 is not essential for virus replication in vitro, it plays an important role in the per os infectivity of AcMNPV in a host-dependent manner. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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10 pages, 3604 KiB

Open AccessArticle

Enhanced Production of Recombinant Protein by Fusion Expression with Ssp DnaB Mini-Intein in the Baculovirus Expression System

by Won Seok Gwak, Jae Bang Choi, Beom Ku Han, Sung Min Bae and Soo Dong Woo

Viruses 2018, 10(10), 523; https://doi.org/10.3390/v10100523 - 25 Sep 2018

Cited by 2 | Viewed by 4214

Abstract

The baculovirus expression system (BES) is considered to be a very powerful tool for the expression of numerous difficult to express vertebrate proteins. Ssp DnaB mini-intein is a useful fusion partner for the production of recombinant proteins because it can be self-cleaved by controlling the pH and temperature, without additional treatment. To evaluate the utility of Ssp DnaB mini-intein in the BES, recombinant viruses were generated to express the enhanced green fluorescent protein, the VP2 protein of porcine parvovirus, and the E2 protein of classical swine fever virus fused to a mini-intein. As expected, intracellular self-cleavage of the mini-intein occurred during virus infection, but the cleavage initiation time varied depending on the target protein. Significantly enhanced protein production was observed for all of the target proteins that were fused to the mini-intein. This increase was enough to overcome the decrease in the fusion protein due to intracellular self-cleavage. The mini-intein in all of the recombinant fusion proteins was successfully cleaved by controlling the pH and temperature. These results suggest that the Ssp DnaB mini-intein is a useful fusion partner in the BES for easy purification and enhanced production of target proteins. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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17 pages, 1410 KiB

Open AccessArticle

Biodiversity, Evolution and Ecological Specialization of Baculoviruses: A Treasure Trove for Future Applied Research

by Julien Thézé, Carlos Lopez-Vaamonde, Jenny S. Cory and Elisabeth A. Herniou

Viruses 2018, 10(7), 366; https://doi.org/10.3390/v10070366 - 11 Jul 2018

Cited by 33 | Viewed by 4845

Abstract

The Baculoviridae, a family of insect-specific large DNA viruses, is widely used in both biotechnology and biological control. Its applied value stems from millions of years of evolution influenced by interactions with their hosts and the environment. To understand how ecological interactions have shaped baculovirus diversification, we reconstructed a robust molecular phylogeny using 217 complete genomes and ~580 isolates for which at least one of four lepidopteran core genes was available. We then used a phylogenetic-concept-based approach (mPTP) to delimit 165 baculovirus species, including 38 species derived from new genetic data. Phylogenetic optimization of ecological characters revealed a general pattern of host conservatism punctuated by occasional shifts between closely related hosts and major shifts between lepidopteran superfamilies. Moreover, we found significant phylogenetic conservatism between baculoviruses and the type of plant growth (woody or herbaceous) associated with their insect hosts. In addition, we found that colonization of new ecological niches sometimes led to viral radiation. These macroevolutionary patterns show that besides selection during the infection process, baculovirus diversification was influenced by tritrophic interactions, explained by their persistence on plants and interactions in the midgut during horizontal transmission. This complete eco-evolutionary framework highlights the potential innovations that could still be harnessed from the diversity of baculoviruses. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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19 pages, 3166 KiB

Open AccessArticle

Establishment of Baculovirus-Expressed VLPs Induced Syncytial Formation Assay for Flavivirus Antiviral Screening

by Shiyu Dai, Yanfang Zhang, Tao Zhang, Bo Zhang, Hualin Wang and Fei Deng

Viruses 2018, 10(7), 365; https://doi.org/10.3390/v10070365 - 11 Jul 2018

Cited by 5 | Viewed by 4407

Abstract

The baculovirus-insect cell expression system has been widely used for heterologous protein expression and virus-like particles (VLPs) expression. In this study, we established a new method for antiviral screening targeting to glycoprotein E of flaviviruses based on the baculovirus expression system. ZIKV is a mosquito-borne flavivirus and has posed great threat to the public health. It has been reported that ZIKV infection was associated with microcephaly and serious neurological complications. Our study showed that either ZIKV E or prME protein expressed in insect cells can form VLPs and induce membrane fusion between insect cells. Therefore, the E protein, which is responsible for receptor binding, attachment, and virus fusion during viral entry, achieved proper folding and retained its fusogenic ability in VLPs when expressed in this system. The syncytia in insect cells were significantly reduced by the anti-ZIKV-E specific polyclonal antibody in a dose-dependent manner. AMS, a thiol-conjugating reagent, was also shown to have an inhibitory effect on the E protein induced syncytia and inhibited ZIKV infection by blocking viral entry. Indeed the phenomenon of syncytial formation induced by E protein expressed VLPs in insect cells is common among flaviviruses, including Japanese encephalitis virus (JEV), Dengue virus type 2 (DENV-2), and tick-borne encephalitis virus (TBEV). This inhibition effect on syncytial formation can be developed as a novel, safe, and simple antiviral screening approach for inhibitory antibodies, peptides, or small molecules targeting to E protein of ZIKV and other flaviviruses. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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16 pages, 2563 KiB

Open AccessArticle

Display of Porcine Epidemic Diarrhea Virus Spike Protein on Baculovirus to Improve Immunogenicity and Protective Efficacy

by Chia-Yu Chang, Wei-Ting Hsu, Yu-Chan Chao and Hui-Wen Chang

Viruses 2018, 10(7), 346; https://doi.org/10.3390/v10070346 - 27 Jun 2018

Cited by 31 | Viewed by 4987

Abstract

A new variant of the porcine epidemic diarrhea virus (PEDV) is an emerging swine disease, killing considerable numbers of neonatal piglets in North America and Asia in recent years. To generate immunogens mimicking the complex spike (S) protein folding with proper posttranslational modification to mount a robust immune response against the highly virulent PEDV, two baculoviruses displaying the full-length S protein (S-Bac) and the S1 protein (S1-Bac) of the virulent Taiwan genotype 2b (G2b) PEDV Pintung 52 (PEDV-PT) strain were constructed. Intramuscular immunizations of mice and piglets with the S-Bac and S1-Bac demonstrated significantly higher levels of systemic anti-PEDV S-specific IgG, as compared with control group. Our results also showed that piglets in the S-Bac group elicited superior PEDV-specific neutralizing antibodies than those of the S1-Bac and control groups. The highly virulent PEDV-PT strain challenge experiment showed that piglets immunized with S-Bac and S1-Bac showed milder clinical symptoms with significantly less fecal viral shedding as compared with non-immunized control piglets. More importantly, piglets immunized with the S-Bac exhibited no to mild clinical signs, with a delayed, minimal viral shedding. Our results demonstrated that the S-Bac could serve as a safe, easy to manipulate, and effective vaccine candidate against the PEDV infection. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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Review

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14 pages, 2486 KiB

Open AccessReview

MultiBac: Baculovirus-Mediated Multigene DNA Cargo Delivery in Insect and Mammalian Cells

by Kapil Gupta, Christine Tölzer, Duygu Sari-Ak, Daniel J. Fitzgerald, Christiane Schaffitzel and Imre Berger

Viruses 2019, 11(3), 198; https://doi.org/10.3390/v11030198 - 26 Feb 2019

Cited by 28 | Viewed by 8297

Abstract

The baculovirus/insect cell system (BICS) is widely used in academia and industry to produce eukaryotic proteins for many applications, ranging from structure analysis to drug screening and the provision of protein biologics and therapeutics. Multi-protein complexes have emerged as vital catalysts of cellular function. In order to unlock the structure and mechanism of these essential molecular machines and decipher their function, we developed MultiBac, a BICS particularly tailored for heterologous multigene transfer and multi-protein complex production. Baculovirus is unique among common viral vectors in its capacity to accommodate very large quantities of heterologous DNA and to faithfully deliver this cargo to a host cell of choice. We exploited this beneficial feature to outfit insect cells with synthetic DNA circuitry conferring new functionality during heterologous protein expression, and developing customized MultiBac baculovirus variants in the process. By altering its tropism, recombinant baculovirions can be used for the highly efficient delivery of a customized DNA cargo in mammalian cells and tissues. Current advances in synthetic biology greatly facilitate the construction or recombinant baculoviral genomes for gene editing and genome engineering, mediated by a MultiBac baculovirus tailored to this purpose. Here, recent developments and exploits of the MultiBac system are presented and discussed. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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12 pages, 2446 KiB

Open AccessReview

Baculovirus as a Tool for Gene Delivery and Gene Therapy

by Chikako Ono, Toru Okamoto, Takayuki Abe and Yoshiharu Matsuura

Viruses 2018, 10(9), 510; https://doi.org/10.3390/v10090510 - 19 Sep 2018

Cited by 55 | Viewed by 11149

Abstract

Based on its ability to express high levels of protein, baculovirus has been widely used for recombinant protein production in insect cells for more than thirty years with continued technical improvements. In addition, baculovirus has been successfully applied for foreign gene delivery into mammalian cells without any viral replication. However, several CpG motifs are present throughout baculoviral DNA and induce an antiviral response in mammalian cells, resulting in the production of pro-inflammatory cytokines and type I interferon through a Toll-like receptor (TLR)-dependent or -independent signaling pathway, and ultimately limiting the efficiency of transgene expression. On the other hand, by taking advantage of this strong adjuvant activity, recombinant baculoviruses encoding neutralization epitopes can elicit protective immunity in mice. Moreover, immunodeficient cells, such as hepatitis C virus (HCV)- or human immunodeficiency virus (HIV)-infected cells, are more susceptible to baculovirus infection than normal cells and are selectively eliminated by the apoptosis-inducible recombinant baculovirus. Here, we summarize the application of baculovirus as a gene expression vector and the mechanism of the host innate immune response induced by baculovirus in mammalian cells. We also discuss the future prospects of baculovirus vectors. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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23 pages, 393 KiB

Open AccessReview

Baculovirus Surface Display of Immunogenic Proteins for Vaccine Development

by Balraj Premanand, Poh Zhong Wee and Mookkan Prabakaran

Viruses 2018, 10(6), 298; https://doi.org/10.3390/v10060298 - 31 May 2018

Cited by 25 | Viewed by 5303

Abstract

Vaccination is an efficient way to prevent the occurrence of many infectious diseases in humans. To date, several viral vectors have been utilized for the generation of vaccines. Among them, baculovirus—categorized as a nonhuman viral vector—has been used in wider applications. Its versatile features, like large cloning capacity, nonreplicative nature in mammalian cells, and broad tissue tropism, hold it at an excellent position among vaccine vectors. In addition to ease and safety during swift production, recent key improvements to existing baculovirus vectors (such as inclusion of hybrid promoters, immunostimulatory elements, etc.) have led to significant improvements in immunogenicity and efficacy of surface-displayed antigens. Furthermore, some promising preclinical results have been reported that mirror the scope and practicality of baculovirus as a vaccine vector for human applications in the near future. Herein, this review provides an overview of the induced immune responses by baculovirus surface-displayed vaccines against influenza and other infectious diseases in animal models, and highlights the strategies applied to enhance the protective immune responses against the displayed antigens. Full article

(This article belongs to the Special Issue Baculovirus Advances and Applications)

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