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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 1359

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 [...] Read more.

(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 1329

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) ( [...] Read more.

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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Restriction endonuclease profiles of the genomic DNA of three different AgMNPV isolates following treatment with HindIII. m denotes the molecular marker. Fragment size in kilobases (Kb) is shown on the left. Red arrowheads indicate restriction fragment length polymorphisms (RFLPs) and asterisks on the left of each lane indicate the presence of submolar bands. Full article ">Figure 2
HindIII restriction endonuclease profiles of the genomic DNA of each of the 18 different AgABB51 genotypic variants (A to R). m denotes the molecular marker. Fragment size in kilobases (Kb) is shown on the left. The profile of genotype A is used as a reference to identify the presence (red arrowheads) or absence (green arrowheads) of characteristic restriction fragments in the other genotypic variants. Genotype A was selected for this purpose because of its similarity to the wild-type isolate (AgABB51) (shown in <a href="#viruses-15-01526-f001" class="html-fig">Figure 1</a>). Full article ">Figure 3
Frequency of AgABB51 genotypic variants A through R. The n-value above each column indicates the number of clones exhibiting each variant’s restriction profile out of a total of 128 clones. Full article ">Figure 4
Mean percentage of mortality caused by AgABB51 wild-type isolate and each of the genotypic variants A–R on A. gemmatalis second instars inoculated with 1.1 × 104 OBs/mL. Error bars indicate the standard error. Different lowercase letters indicate significant differences between variants (ANOVA, Tukey HSD; p < 0.05). Full article ">Figure 5
(a) Median time to death (MTD) values for the AgABB51 isolate and selected genotypic variants in A. gemmatalis second instars. Error bars indicate standard error and different lowercase letters indicate significant differences between variants (Bonferroni-adjusted t-test; p < 0.05). (b) OB production values obtained for AgABB51 and the selected genotypic variants in A. gemmatalis fifth instars. Error bars indicate standard error and different lowercase letters indicate significant differences between variants (Tukey HSD test; p < 0.05). Full article ">

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

Viewed by 2675

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, [...] Read more.

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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Figure 1
Amplification plots for the qPCR evaluation of different baculovirus concentrations in the range of 0 (i.e., blank)−1.1 × 107 IU/mL (A) as well as the linear regression over the concentration range (B). The number of replicate measurements was n = 39 for 1.1 × 102 IU/mL and 1.1 × 107 IU/mL and n = 18 for all other concentrations and the blank. Full article ">Figure 2
Amplification plots of the baculovirus DNA by qPCR measurements. The DNA was extracted from samples taken either from the supernatant or from the cell pellet of infected cells after 5 min (i.e., 0 h) of incubation and compared to a negative control (A). The amount of viral DNA detectable in the cell pellet was monitored over the course of 7 h (B) and compared to values after 18 h of infection (C). All samples were prepared in triplicates, with error bars only being displayed in (A) to allow a clearer view of the data. Full article ">Figure 3
Development of the flow cytometric quantification protocol for baculoviruses (either wild-type or virus expressing the green fluorescent protein, BV-wt and BV-GFP, respectively). In a first approach, (A) the detection capability for both viruses was determined without further method optimization after 18 h of cell infection and subsequent flow cytometric detection of fluorescent cells. Depending on the staining procedure, green (no staining, left column), yellow (PE staining, middle column), or red fluorescence (APC staining, right column) was evaluated. The infection kinetics of the three approaches (B) indicate the earliest possible time of quantification at which the linear calibration range was subsequently determined for each individually optimized strategy (C). Error bars depict standard deviation of technical triplicates (A,B) and 18 replicates with n = 6 on three different days for (C). Full article ">Figure 4
Quantification of the percentage of fluorescing cells after BV infection and fluorescent labeling of the mRNA of the viral gp64 protein within the first four hours of infection. A multiplicity of infection (MOI) of 1 was used and compared to a negative control without virus infection (error bars indicate the standard deviation of technical triplicates). Full article ">

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 1564

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) [...] Read more.

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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Reprogramming chiA and v-cath transcription. (a) Schematic depicting the method used for switching the AcMNPV chiA/v-cath intergenic promoters via an EGFP-selectable virus (AcEGFP) as described before [<a href="#B19-viruses-15-00503" class="html-bibr">19</a>]. The small arrows indicate which promoter (wt, p6.9, or polh) is driving chiA or v-cath transcription. To the right is a summary of the chiA and v-cath expression from each virus (+ = expressed, − = not expressed). (b) Intergenic chiA/v-cath promoter sequence of AcMNPV, AcMNPV-Rep and the dual reprogrammed virus (Acp6.9-chiA/polh-cath). The chiA and v-cath translation start codons are bolded and restriction enzyme recognition sequences (cloning sites) are italicized. For the dual reprogrammed virus, the sequence of the p6.9 promoter (driving chiA) is shown from 3′-5′ and that of the adjacent polh promoter (driving v-cath, shaded in grey) are shown from 5′-3′ to reflect the coding strand for each antiparallel ORF. Potential mRNA transcription sites (TAAG) for chiA (dark grey) or v-cath (white) are boxed. The indicated chiA transcription site was mapped for AcMNPV and Acp6.9-chiA [<a href="#B19-viruses-15-00503" class="html-bibr">19</a>], but not Acp6.9-chiA/polh-cath. The v-cath transcription site was mapped previously [<a href="#B20-viruses-15-00503" class="html-bibr">20</a>]. Full article ">Figure 2
Temporal CHIA and proV-CATH expression by AcMNPV-Rep, Acp6.9-chiA, and Acp6.9-chiA/polh-cath in infected SF-21 cells. (a) Northern blots of chiA and v-cath mRNAs over a time-course of infection of SF-21 cells with AcMNPV-Rep, Acp6.9-chiA, and Acp6.9-chiA/polh-cath. Sizes are based on a high-range RNA ladder (Fermentas). Transcripts were detected using DIG-labeled ssDNA probes as described in [<a href="#B19-viruses-15-00503" class="html-bibr">19</a>]. Ethidium bromide-stained rRNA bands indicate RNA equivalency (10 µg/lane). Lane M is an uninfected control sample. (b) Temporal CHIA and proV-CATH production (0–48 h p.i.). Total proteins isolated from SF-21 cells infected with the indicated virus were loaded (in equivalent volumes) on gels for protein blots and probed with either anti-BmCHI-h antibody (to detect CHIA), anti-V-CATH antibody (to detect proV-CATH), or anti-GAPDH antibody (to detect host GAPDH) as a loading control. Lane M is protein from uninfected cells as control. Lane G is protein from AcEGFP-infected cells. (c) CHIA and proV-CATH solubility. Lysates from infected cells were separated by centrifugation into detergent (0.5% NP-40/1% Triton-X)-soluble (SOL) and -insoluble (INSOL) fractions and proteins were detected with anti-BmCHI-h or anti-V-CATH antibody. Full article ">Figure 3
Chitinase and cathepsin (protease) assays. Homogenates from infected SF-21 cells were used to compare viral chitinase (CHIA) and protease (V-CATH) activities at 48 h p.i. with AcMNPV-Rep, Acp6.9-chiA, and Acp6.9-chiA/polh-cath. Mock-infected (Mock) and AcEGFP-infected cells were used as negative controls. (a) Chitinase assay. Total protein (50 µg) was used to measure viral CHIA activity. (b) Protease assays. Total protein (400 µg) was used to measure V-CATH activity in the presence or absence of 20 µM of the cysteine protease inhibitor E-64. Full article ">Figure 4
Budded virus production of AcEGFP-, AcMNPV-Rep-, Acp6.9-chiA-, and Acp6.9-chiA/polh-cath-infected cells. SF-21 cells were infected (m.o.i. = 0.1 PFU/cell), and at the indicated timepoints, a small amount (0.2 mL) of supernatant was removed and titrated by end-point dilution. The data points represent the mean budded virus titers from three replicates. Error bars denote standard deviations. Full article ">Figure 5
Temporal CHIA and V-CATH protein production from infected T. ni larvae. (a) Carcass. CHIA (50 µg/lane) and V-CATH/proV-CATH (500 µg/lane) were immunodetected from homogenates of infected larvae collected at 3 and 4 days p.i., as indicated, using anti-BmCHI-h antibody (to detect CHIA) and anti-V-CATH antibody (to detect proV-CATH). (b) Hemolymph. CHIA and V-CATH/proV-CATH were immunodetected in pooled (3 larvae each) hemolymph samples extracted at 3 days p.i. The same volume of hemolymph (15 µL) from virus-infected larvae were loaded in each lane. Migration of protein markers is shown on the right in kDa. Full article ">Figure 6
Chitinase and cathepsin (protease) assays from infected T. ni larval tissues. Total protein in larval homogenates was used to compare viral chitinase (CHIA) and protease (V-CATH) activities at 3, 4, and 5 days p.i. for AcEGFP, AcMNPV-Rep, Acp6.9-chiA, and Acp6.9-chiA/polh-cath. (a) Total protein (200 µg) was used to measure viral chitinase (CHIA) activity. ns, not significant (b) Protease assays. Total protein (400 µg) was used to measure V-CATH activity in the presence (white bars) or absence (black bars) of E-64 (20 µM) inhibitor. Statistics were computed only for V-CATH samples without E-64. The indicated p-values (t-test, 95% CI) were ≤ 0.001 (*), ≤ 0.002 (**), and ≤ 0.003 (***). Full article ">Figure 7
Survival of infected fifth instar T. ni larvae. Larvae were injected with 10 µL containing 5 × 104 TCID50 units of each virus and were monitored every 8 h for viability at 27 °C. About thirty insects per experiment were injected with each virus and monitored for viability by prodding with a blunt object. The survival curves combine data from three independent experiments. The data were analyzed using the Kaplan–Meier method, and the survival curves were found to be significantly different from each other by Log-rank test (p < 0.0001). The dashed horizontal line indicates 50%. Error bars indicate standard error. Full article ">Figure 8
Pathology of infected fifth Instar T. ni larvae. Larvae were injected with 10 µL containing 5 × 104 TCID50 units of each virus and incubated at 27 °C in individual containers with synthetic diet. They were monitored every 24 h for pathological changes (i.e., live, dead, and liquefied). (a) Insects with characteristic phenotypes due to infection with each of the viruses were photographed at 4 and 5 days as indicated. (b) The graphed results combine data for 3 to 6 days p.i. for three independent experiments. Error bars indicate standard deviation. Full article ">

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 1557

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 [...] Read more.

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 2300

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 [...] Read more.

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 1892

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 [...] Read more.

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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Figure 1
Flowchart of constructing AcMNPV genome containing modified C1 fragment. (A) Circular maps of the parental genome of AcMNPV-WIV-Syn1 and the modified AcMNPV-Syn-mC1-1.1. (B) The design-build-test cycling method to modify C1 fragment. The design of the modified C1 was based on the modification of B1, B2, and B3 fragments. Non-essential genes of each fragment were removed to generate modified fragments of mB1-1.0, mB2-1.0, and mB3-1.0. mB-1.0, mB2-1.0, and mB3-1.0 by overlapping PCR and used to generate modified genomes of AcMNPV-Syn-mB1-1.0, AcMNPV-Syn-mB2-1.0 and AcMNPV-Syn-mB3-1.0, respectively, using two steps of TAR in yeast. Meanwhile, AcMNPV-Syn-mC1-1.0 was generated using mB1-1.0, mB2-1.0, and mB3-1.0. To test, transfection and infection were performed; and the modified genome, which could not generate efficient progeny viruses, was redesigned and subjected to the design-build-test cycle again. Full article ">Figure 2
Design and characterization of version 1.0 of the modified genomes. (A) Design of mB1-1.0, mB2-1.0, and mB3-1.0. The modified fragments are shown on the backbones of the original B1, B2, and B3. The deleted genes and hrs are coloured white, the reserved genes green, and the reserved hr1a blue. The primers used for overlap extension PCR are indicated. (B) Transfection and infection results of AcMNPV-Syn-mB1-1.0, AcMNPV-Syn-mB2-1.0, AcMNPV-Syn-mB3-1.0, and AcMNPV-Syn-mC1-1.0. The images were obtained at 96 h post-transfection (p.t.) and 96 h post-infection (p.i.). The green dots represent infected Sf9 cells. Full article ">Figure 3
Redesign and characterization of AcMNPV-Syn-mB1-1.1, AcMNPV-Syn-mB2-1.1, and AcMNPV-Syn-mC1-1.1. (A) Design of mB1-1.1 and mB2-1.1. The modified fragments are shown on the backbones of the original B1 and B2. The colouring is similar to that in <a href="#viruses-14-02780-f002" class="html-fig">Figure 2</a>A, with the eight repaired ORFs showed in brown. (B) Transfection and infection results of AcMNPV-Syn-mB1-1.1, AcMNPV-Syn-mB2-1.1, AcMNPV-Syn-mC1-1.1, and AcMNPV-WIV-Syn-1. The images were obtained at 96 h p.t. and 96 h p.i. The green dots represent infected Sf9 cells. Full article ">Figure 4
Restriction enzyme analyses of the genomes of AcMNPV-Syn-mC1-1.1 and AcMNPV –WIV-Syn1. (A) Physical maps of the AcMNPV-Syn-mC1-1.1 and AcMNPV-WIV-Syn1 genomes digested with BglII, XhoI or SacII. The fragments were named alphabetically according to size. The fragments corresponding to the C1 region in AcMNPV-WIV-Syn1 and the modified C1 region in AcMNPV-Syn-mC1-1.1 are highlighted. (B) Computer-simulated restriction enzyme profiles of AcMNPV-WIV-Syn1 and AcMNPV-Syn-mC1-1.1. (C) Electrophoresis results of the restriction enzyme-digested DNA samples of AcMNPV-WIV-Syn1 and AcMNPV-Syn-mC1-1.1 in 1% agarose gel. Fragments with size change are indicated in red letters in AcMNPV-Syn-mC1-1.1 and the original fragments in AcMNNPV-WIV-Syn1 are marked with a red star. Full article ">Figure 5
Characterization of AcMNPV-Syn-mC1-1.1. (A) One-step growth curves of AcMNPV-WIV-Syn1 and AcMNPV-Syn-mC1-1.1. Sf9 cells were infected with AcMNPV-WIV-Syn1 or AcMNPV-Syn-mC1-1.1 at an MOI of 1, the supernatants were collected at the indicated time points and viral titres were titrated by EPDA. The experiment was carried out in triplicate, and the titre was transformed logarithmically after averaging. Statistical significance was determined by 2way ANOVA. ***: p < 0.001. (B) Results of electron microscopy. Sf9 cells were infected with AcMNPV-WIV-Syn1 or AcMNPV-Syn-mC1-1.1 at an MOI of 5, and cells were collected at 24, 48, and 72 h p.i. for electron microscopy. The representative nucleocapsids are marked with arrows. OB: occlusion body. Scale bars are 2 μm and 1 μm. Full article ">

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 4 | Viewed by 2143

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 [...] Read more.

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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Figure 1
The promoters included on commercially available transfer plasmids have drastically different transcription profiles. (Left panel). The transcript abundance of AcMNPV polh, p10, gp64, and ie1 ORFs, which are among the only promoters available on commercial transfer plasmids for foreign gene expression. (Right panel). The transcript abundance profiles of AcMNPV ORFs selected for evaluation of the upstream promoter regions in this study. Promoters were selected for expression profiles between polh/p10 and gp64/ie1. Full article ">Figure 2
Production of intracellular GFP from selected AcMNPV promoters. (A) Median fluorescence intensity measured using flow cytometry and (B) relative transcript abundance measured using RT-qPCR at various times post infection. Full article ">Figure 3
Production of extracellular SEAP from selected AcMNPV promoters. (A) Yield of SEAP (mg/L) of culture supernatants measured using a colorimetric SEAP activity assay and (B) relative transcript abundance measured using RT-qPCR at 24, 48, and 72 h post infection. Full article ">Figure 4
Evaluation of the 5<math display="inline"><semantics> <msup> <mrow/> <mo>′</mo> </msup> </semantics></math>UTRs of AcMNPV ORFs categorized according to transcript abundance. (A) Length (in nucleotides) and (B) A/T content of the 5<math display="inline"><semantics> <msup> <mrow/> <mo>′</mo> </msup> </semantics></math>UTR between the late gene promoter motif (5<math display="inline"><semantics> <msup> <mrow/> <mo>′</mo> </msup> </semantics></math>-TAAG-3<math display="inline"><semantics> <msup> <mrow/> <mo>′</mo> </msup> </semantics></math>) and translation initiation codon (ATG). * p < 0.05, ** p < 0.01. Full article ">Figure 5
Circular chromosome map of AcMNPV ORFs colour-coded according to transcript abundance. Full article ">Figure 6
Distance (in nucleotides) between the start codon and 5<math display="inline"><semantics> <msup> <mrow/> <mo>′</mo> </msup> </semantics></math> end of each homologous region for every AcMNPV ORF categorized according to transcript abundance. The distance was calculated by subtracting the genomic location of the 5<math display="inline"><semantics> <msup> <mrow/> <mo>′</mo> </msup> </semantics></math> end of each AcMNPV ORF from the genomic location of the 5<math display="inline"><semantics> <msup> <mrow/> <mo>′</mo> </msup> </semantics></math> end of each hr. Positive values represent ORFs located behind (clockwise) to the hr and negative values represent distances between ORFs that are located in front of (counterclockwise) the hr. Full article ">Figure 7
The approximate positions of the upstream octamer (blue) and downstream octamer (red) in regions upstream of the late gene promoter motif for the most abundant AcMNPV ORFs. Full article ">Figure 8
Consensus sequences calculated from multiple sequence alignments. (A) Consensus sequence for the nucleotide sequences flanking the translation initiation site and (B) the late gene promoter motif. Consensus sequences were calculated from multiple sequence alignments of sequences extracted from AcMNPV ORFs that were categorized according to transcript abundance. Full article ">

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 1581

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, [...] Read more.

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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Figure 1
BmHEXIM1 showed a significant response to BmNPV infection. (A) The expression levels of BmHEXIM1 in BmN cells at different hours post infection with BmNPV. BmN cells without BmNPV infection as the control. (B) The expression levels of BmHEXIM1 in different tissues of silkworm post infection with BmNPV. Fat body of silkworm without BmNPV infection as the control. (C) Time course analysis of subcellular localization of BmHEXIM1 by immunofluorescence in BmN cells infected by BmNPV; DAPI, blue; and BmHEXIM1, green. The mRNA level of target genes was normalized to the internal control (BmRPL32). Data represent mean ± SEM of the three independent experiments. The number of asterisks represents the degree of significance with respect to p-value. P-values were provided as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Full article ">Figure 2
RNAi down-regulated BmHEXIM1 and inhibited the invasion of BmNPV. (A) The RNAi of the siBmHEXIM1 RNA decreases the expression level of protein HEXIM1 at 48 h post-transfection. (B) TCID50 end-point dilution was used to evaluate the production of infectious BV at 24 h and 48 h. (C) The infected cells (the cells with the green fluorescence) were observed by an inverted fluorescence microscope at 24 h and 48 h (bar = 100 μm). (D) BmN cells were transfected with si-NC and siBmHEXIM1 RNA for 48 h. Then, the cells were infected with BmNPV for 48 h. The viral gene’s expression was measured by qRT-PCR analysis. (E) The BmNPV abundance was assessed by analyzing the expression of Bm14 by Western blot after knockdown of BmHEXIM1. The mRNA level of target genes was normalized to the internal control (BmRPL32). Data represent mean ± SEM of the three independent experiments. The number of asterisks represents the degree of significance with respect to p-value. p-values were provided as * p < 0.05; ** p < 0.01; **** p < 0.0001. Full article ">Figure 3
BmHEXIM1 promoted BmNPV proliferation, and its full length was essential to promote viral transcription. (A) BmN cells were transfected with pIZ/V5-BmHEXIM1 expressing plasmid, and the expression of BmHEXIM1 was analyzed by Western blot at 24 h, 48 h, 72 h, and 96 h. (B) TCID50 end-point dilution was used to evaluate the production of infectious BV at 24 h and 48 h. (C) The infected cells (the cells with green fluorescence) were observed by an inverted fluorescence microscope at 24 h and 48 h (bar = 100 μm). (D) Determination of the region of BmHEXIM1 promoted BmNPV proliferation. (E) BmN cells were transfected with pIZ/V5-BmHEXIM1, pIZ/V5-BmHEXIM1-1, and pIZ/V5-BmHEXIM1-2 expressing plasmid for 48 h. Then, the cells were infected with BmNPV for 48 h. The viral gene’s expression was measured by qRT-PCR analysis. (F) BmNPV abundance was assessed by analyzing the expression levels of Bm14 by Western blot. The mRNA level of target genes was normalized to the internal control (BmRPL32). Data represent mean ± SEM of the three independent experiments. The number of asterisks represents the degree of significance with respect to p-value. P-values were provided as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Full article ">Figure 4
BmHEXIM1 suppresses host innate immunity during BmNPV infection. (A–D) Quantification of the expression of BmSTING, BmRelish, BmcecA, and BmcecB by qRT-PCR after overexpression of BmHEXIM1, BmHEXIM1-1, BmHEXIM1-2, or knockdown of BmHEXIM1 in BmN cells after BmNPV infection. (E) The phosphorylation level of RNP Ⅱ by Western blot after overexpression of BmHEXIM1, BmHEXIM1-1, BmHEXIM1-2, or knockdown of BmHEXIM1 in BmN cells. (F–I) In the absence or presence of PMA, the expression of BmSTING, BmRelish, BmcecA, and BmcecB was also analyzed by qRT-PCR or by the co-expression of HEXIM1. The mRNA level of target genes was normalized to the internal control (BmRPL32). Data represent mean ± SEM of the three independent experiments. The number of asterisks represents the degree of significance with respect to p-value. p-values were provided as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Full article ">Figure 5
HMBA inhibited BmRelish-mediated pathway and promoted BmNPV proliferation. (A) BmN cells were stimulated with increasing concentrations of HMBA (1, 5, 10, 20, and 30 mM) for 24 h, and the expression levels of BmHEXIM1 were measured by qRT-PCR analysis. (B) BmN cells stimulated or not with HMBA (10 mM) for 12 h, 24 h, and 48 h and the expression of BmHEXIM1 were measured by qRT-PCR analysis. (C) BmN cells stimulated with HMBA (10 mM) for 24 h and the expression of viral genes were measured by qRT-PCR analysis. (D) BmN cells stimulated with HMBA (10 mM) for 24 h, and then added BmNPV for 48 h. BmNPV abundance was assessed by analyzing the expression of Bm14 by Western blot. (E) BmN cells stimulated with HMBA (10 mM) for 24 h and the expression levels of BmSTING, BmRelish, BmcecA, and BmcecB were measured by qRT-PCR analysis. The mRNA level of target genes was normalized to the internal control (BmRPL32). Data represent mean ± SEM of the three independent experiments. The number of asterisks represents the degree of significance with respect to p-value. p-values were provided as ** p < 0.01; *** p < 0.001; **** p < 0.0001. Full article ">Figure 6
Schematic representation of the putative mechanism(s) of BmHEXIM1 in assisting BmNPV evade antiviral immune response and promoting BmNPV proliferation. (A) BmNPV significantly induced BmHEXIM1 expression, while activating host antiviral pathways, such as STING and Relish. The innate immune pathway mediated by BmRelish can induce the expression of BmSTING antimicrobial peptide genes (BmcecA and BmcecB) to resist BmNPV infection. (B) Altered subcellular localization of BmHEXIM1 may be regulated by ubiquitination following viral infection. “? “ were provided as “not determined”. (C) BmHEXIM1 inhibits BmRelish-dependent transcription, leading to suppression of the host’s antiviral response. (D) Stimulation of PMA inhibits BmHEXIM1 to repress BmRelish-dependent transcription. (E) HMBA treatment induces BmHEXIM1 expression to suppress host antiviral response. Full article ">

Review

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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 1427

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. [...] Read more.

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 4168

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 [...] Read more.

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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