Autographa Californica Nuclear Polyhedrosis Virus Research Articles

Effects of wasp (Vespa crabro) nest extracts on virus replication of Autographa californica nuclear polyhedrosis virus on Spodoptera frugiperda cell culture.

The online version contains supplementary material available at 10.1007/s10616-023-00603-0.

Spodoptera frugiperda Hücre Kültüründe Geliştirilen Autographa californica nükleer polihedrosis virüsünün (AcNPV) Replikasyonu Üzerine Türkiye Propolisinin Antiviral Etkisi

In this study, the influence of propolis extracts gathered from Turkey on the replication of Autographa californica nuclear polyhedrosis virus (AcNPV) developed in Spodoptera frugiperda culture was researched through examining changes in cytopathic influences, hydroxyl radical mediated DNA damage and progeny virus concentration. By making use of propolis extract, we found inhibiting effect on cytopathic changes and DNA damage. It was found that propolis extracts on the cytopathic changes and hydroxyl radical-mediated DNA damage were similar to the control. The results showed that propolis has a protective effect on DNA exposed with H2O2. In terms of the production of occlusion bodies in Sf9 cells, no important difference was found between the average percentage of contaminated cells in contaminated control cells when the concentration of propolis is 3.125, and 1.5625 μg/mL (4.018 (Plaque forming units (PFU) pfu/mL and 5.145 pfu/mL respectively) (p<0.05) and no significant difference was found between the average number of occlusion bodies in the contaminated control cells and the contaminated cells in the concentration of propolis. Respectively 98.70% and 98.67% reduction were found in virus titter with propolis extracts when the concentration of propolis extracts 100, 50 μg/mL were included 1 h p.i. (p<0.05). However, different concentrations of propolis extract reduced the number of progeny viruses significantly.

Chrysalises as natural production units for recombinant subunit vaccines

The baculovirus vector expression system (BEVS) combines cultured insect cells and genetically modified Autographa californica nuclear polyhedrosis virus (AcMNPV)-derived baculovirus vectors. This expression system has been widely used for the expression of hundred of proteins for more than 30 years, existing commercial products manufactured at large scale by this methodology, mainly subunit vaccines. At an industrial scale, insect cells, as any other cultured cells, require artificial media and a strict control of environmental sterile conditions in the complex and expensive bioreactors. Here we describe an efficient alternative to produce recombinant biologics using the versatile and productive baculovirus vectors. It consists in natural biocapsules (pupae from Trichoplusia ni (Hübner) Lepidoptera), containing millions of insect cells in perfect physiological conditions, ready to be programmed by a genetically modified AcMNPV-derived baculovirus vector to produce large quantities of any recombinant protein. This technology, denominated CrisBio, has been tested to produce dozens of proteins, reaching productivities on the range of milligrams per infected pupa, that can be translated into dozens of vaccine doses, for example. The biologics production by CrisBio was industrialized with the design of both insect rearing and pupae storage single-use plastic devices, compatible with machines specifically designed for the automation of pupae manipulation and inoculation. These devices and machines reduce manual operations, increase batches consistency and facilitate the scaled production of any recombinant protein. As a mode of examples, the productivity in CrisBio technology platform of two virus-like particle (VLP) vaccine antigens is described in this work.

Overexpression of a virus-like particle influenza vaccine in Eri silkworm pupae, using Autographa californica nuclear polyhedrosis virus and host-range expansion.

Ecological investigations of silkworms have revealed that Eri silkworms (Samia cynthia ricini) possess useful morphological and ecological characteristics for virus-like particle (VLP) production, namely non-seasonal breeding, longer lengths, and heavier weights than Bombyx mori silkworms. Furthermore, when vector DNA from Bombyx mori nuclear polyhedrosis virus (BmNPV), which is unable to replicate in Sf9 cells from Eri silkworms, was replaced with the Autographa californica nuclear polyhedrosis virus (AcNPV) vector, three improved AcNPV influenza virus recombinants capable of replication in Sf9 cells were obtained. Although VLP antigens produced previously in silkworms were not evaluated individually, the present recombinant Fukushima (FkH5) and Anhui (AnH7) VLP antigens were detected in tissue fluids and fat bodies of Eri silkworms. Here, we aimed to determine the function of the AcNPV vector and P143 gene by expressing recombinants in Sf9 cells and eri silkworm pupae. The FkH5 recombinant produced high yields of haemagglutinin (HA)-positive VLPs, showing a mean HA titre of 1.2 million. Similarly, high production of H7 HA VLPs was observed in the fat bodies of eri silkworm pupae. Antigenic analysis and electron microscopy examination of Eri-silkworm-produced H5 HA VLPs showed characteristic antigenicity and morphology similar to those of the influenza virus. Although FkH5 recombinants possessing the AcNPV vector did not replicate in Bm-N cells, the introduction of the helicase p143 gene from BmNPV resulted in their production in Bm-N and Sf9 cells.

Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus

Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 107 pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 105 egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases.

이산화염소의 활성산소 생성 유도에 의한 항암 및 항바이러스 활성

Chlorine dioxide has been used for a disinfectant by exhibiting antimicrobial activity and is also potent to kill insect pests infesting stored grains. This study aimed to extend the usefulness of chlorine dioxide with respect to anticancer and antiviral activities. Cytotoxicity of chlorine dioxide was assessed against five different human cancer cell lines. Chlorine dioxide exhibited significant cytotoxicity against two breast cancer cell lines (MCF-7, MDA-MB-231) and three colorectal cancer cell lines (LoVo, HCT-116, SW-480). This cytotoxicity appeared to be associated with the capacity of chlorine dioxide to induce the production of reactive oxygen species (ROS). Compared to control insect cell lines, the cancer cell lines possessed much higher levels of ROS. On the other hand, a treatment of an antioxidant, vitamin E, significantly reduced the cytotoxicity, suggesting that the cytotoxicity was induced by high levels of ROS production. Chlorine dioxide exhibited antiviral activity against different viruses. A baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), is a dsDNA insect virus and lost its viral activity to form polyhedral viral particles in response to chlorine dioxide. The antiviral activity against AcNPV was dependent on the incubation time with chlorine dioxide. Tobacco mosaic virus is a ssRNA plant virus and was reduced in its population after exposure to chlorine dioxide along with significant decrease of viral symptoms. These results indicate that chlorine dioxide possesses anticancer and antiviral activities probably due to its inducing activity of ROS production.

Establishment and characterization of a novel cell line from midgut tissue of Helicoverpa armigera (Lepidoptera: Noctuidae).

The midgut of lepidopteran larvae serves as a target for many pathogens such as Bacillus thuringiensis (Bt). Cell lines originating from midgut tissues will be very helpful tools in many research fields. However, to date, no Bt-susceptible midgut-derived cell lines are available. Here, we reported that a novel cell line, designated as HNU-Ha-MG1, was established from midgut tissue of the fourth instar larvae of Helicoverpa armigera. This cell line grew well in Grace's insect cell culture medium supplemented with 10-15% fetal bovine serum. The shape of the most cells was round or polygonal, and some tended to aggregate to form multiple cell masses. The size of the cells was 13.8 ± 1.8μm in diameter, and the maximum density reached (2.40 ± 0.15) × 10(6) cells/ml. The population doubling time during logarithmic growth phase was 58.6 ± 7.0h at 28°C. The number of chromosomes was about 90-130, which exhibited typical chromosome characteristics of lepidopteran cell lines. The patterns of random amplified polymorphic DNA of the cell line were different from those of Sl-HP and Hi5 cell lines which were frequently used in our laboratory. 20-Hydroxyecdysterone induced apoptosis in a very small part of cells at 2μg/ml but did not affect expression of autophagy-related protein 8 (Atg8) and its lipidation at 36h post-treatment. The cell line was permissive to Autographa californica nuclear polyhedrosis virus (AcMNPV) and H. armigera nuclear polyhedrosis virus (HaSNPV). This cell line was found to be susceptible to activated Cry1C at the final concentration of 0.5-1.0μg/ml but not to the activated Cry1Ac.

Screening of silkworm strains for efficient recombinant protein production by Autographa californica nucleopolyhedrosis virus (AcNPV)

Baculoviruses base vectors come to be regarded as methods for in vivo gene delivery and transient expression to the silkworm. In the case of silkworm, B. mori, two types of baculoviruses, AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV (Bombyx mori nuclear polyhedrosis virus), are potentially applicable as vectors. Recently, AcNPV showed promising results with some silkworm strains despite different hostspecificities. We searched for a highly-permissive silkworm strain in the B. mori stocks of Kyungpook National University that could produce high levels of recombinant protein. Seventy strains were screened using the recombinant AcNPV/BmA3-Luc virus. Based on the measured luciferase activity, the strains could be divided into three groups, high-, middle-, and low-permissive strains, according to their relative recombinant protein expression levels. At 48 hours post-injection, the luciferase activity in the high-permissive strains was 500-fold greater than that of the low-permissive strains. At 72 hours post-injection, a significant elevation in luciferase activity was observed in the hemocytes of all strains. Then, based on the above results, the High Permissive Strain (HPS) S10 and the Low Permissive Strain (LPS) S39 were pick up and was carried out Dot blotting, RT-PCR and Real time PCR.

Dimerization and proper degradation of BmNPV IE2 are required for efficient virus growth in larvae of Bombyx mori (Lepidoptera: Bombycidae)

The baculovirus ie2 gene is one of the immediate early genes, and its product is known to transactivate viral promoters. However, the roles of Bombyx mori nucleopolyhedrovirus (BmNPV) ie2 in insect larvae are poorly understood. Here we investigated the functions of BmNPV IE2 in cultured cells and in insect larvae using two mutant viruses, BmIE2D and BmIE2CS. BmIE2D lacks the IE2 C-terminal coiled-coil domain that is required for IE2 dimerization. The other mutant BmIE2CS expresses an E3 ligase activity-deficient IE2 derivative, which is degraded more slowly compared with wild-type IE2. We found that ie2 mutations had little effect on BmNPV infection in cultured cells, whereas budded virus and occlusion body production was significantly reduced in the hemolymph of B. mori larvae infected with ie2 mutants. These results indicate that both dimerization and proper degradation of BmNPV IE2 are crucial steps for efficient virus growth in B. mori larvae, but not in cultured cells. Oral infection assays also revealed that the infectivity of the occluded form of ie2 mutants was normal in B. mori larvae, which is inconsistent with the results reported from ie2 mutants of Autographa californica NPV. This suggests that loss of IE2 function causes virus-specific effects in host insects.

Development of a novel baculovirus titration method using the Enzyme-linked immunosorbent spot (ELISPOT) assay

The baculovirus expression vector system (BEVS) is one of the most powerful methods for production of recombinant proteins for research or commercial purposes. Titration of viable virus in insect cell culture is often required when BEVS is used for basic research or bioprocessing. An enzyme-linked immunosorbent spot (ELISPOT) assay using monoclonal antibodies against the major capsid protein VP39 of both Autographa californica nuclear polyhedrosis virus (AcMNPV) and Bombyx mori nuclear polyhedrosis virus (BmNPV) was developed for baculovirus quantitation at 48h post-infection. The titer was determined by visualizing infected insect cells as blue spots and automated spot counting was achieved with ELISPOT hardware and software. Log-scale comparison of the results between the ELISPOT assay and a conventional end point dilution assay using a fluorescent marker showed a good correlation for both AcMNPV (R2=0.9980, p<0.05) and BmNPV (R2=0.9834, p<0.05). In conclusion, a novel, rapid and semi-automated procedure for titrating baculovirus was developed based on the specific immunostaining of infected cells followed by automated spot counting.

The Effect of a Small Conotoxin-Like ctx Gene from Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) on Insect Hemolymph Melanization

The conotoxin-like (ctx) gene encodes a small cysteine-rich polypeptide in various baculoviruses. Previous research has demonstrated that the product of the ctx gene could be purified from insect cells infected by Autographa californica nuclear polyhedrosis virus (AcMNPV), but its function was unknown. In this paper, we compared the conserved cysteine motif structure (CX3GX2CX5CCX3CX6C) of the ctx gene in baculoviruses and generated recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV) with the BmNPV bacmid system. The recombinant BmNPV contained the ctx gene from AcMNPV or a fusion gene of ctx with eGFP, respectively. Fluorescence in CTX-eGFP-positive cells was mainly observed on the cell membrane. To gain insight into CTX function, two methods were used to elucidate the affect CTX had on hemolymph melanization in vivo and in vitro in insect larvae and pupae. The results indicated that CTX abrogates hemolymph melanization; however, the mechanisms require further evaluation.

Modulation of innate immunity in chickens induced by in vivo administration of baculovirus

Baculoviruses stimulate cytokine production in mammalian cells. They induce a strong innate immune response in animals and have adjuvant properties. The purpose of this work was to study the in vivo effect of baculovirus on chicken innate immune response. SPF chickens were inoculated intravenously with Autographa californica nuclear polyhedrosis virus (BV). Three hours later, chickens were bled, euthanized and their spleen, duodenum and cecal tonsils were excised in order to take samples for RNA extraction and real time PCR, and to isolate lymphocytes, which were stained and analyzed by flow cytometry. The results obtained showed that baculovirus inoculation up-regulates the expression of IFN-γ, IL-6 and LITAF in spleen cells. This result (IFN-γ) correlated with that obtained by ELISA which showed a very strong increase of IFN-γ in chicken plasma. Flow cytometry analysis revealed that BV inoculation induced in spleen an increase in the percentage of monocyte/macrophage population together with an increase in CD3+CD4+ T lymphocytes. On the other hand, BV inoculation decreased the percentage of CD3+CD4+ T lymphocytes and increased the percentage of NK cells in cecal tonsils. However, intraepithelial lymphocytes of the gut did not show differences between BV and control treated animals. Even though further studies in order to understand the mechanisms by which BVs affect the avian immune response are needed, results obtained in the present work demonstrate the ability of BVs to stimulate the innate immunity in chickens, modifying the expression pattern of related genes and the profile of the immune cells involved.

Secretory expression of all 16 subtypes of the hemagglutinin 1 protein of influenza A virus in insect cells

Influenza viruses are known for their ability to change their antigenic structure and create new viral strains. Hemagglutinin (HA), for which 16 subtypes have been identified, is a major antigenic determinant essential for the pathogenesis of influenza A viruses. To predict and monitor future epidemics, it is critical to produce various HA subtype antigens conveniently and rapidly. A simple, effective, and economic method to generate subunit HA1 of all 16 HA subtypes as recombinant proteins (rHA1) is reported. rHA1 proteins are expressed in insect cells as secretory proteins after integration into a baculovirus expression vector containing a 6 × His tag element and the signal peptide of the GP67 protein, a membrane glycoprotein identified in Autographa californica nuclear polyhedrosis virus. The proteins can be purified to ≥90% purity using a single Ni 2+-chelating affinity chromatography step, yielding a recovery rate of about 50%. The rHA1 proteins elicit high titer antibodies in mice and show high specificity in Western blots. This study paves the way for subtype specific detection methods and for future studies of the immune relationships among the subtypes of influenza A virus HA proteins.

Establishment, growth kinetics, and susceptibility to AcMNPV of heat tolerant lepidopteran cell lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33°C and 35°C was carried out by shifting the culture temperature between 28°C and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28°C, 33°C, 35°C and 37°C, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.

Phylogenetic Relationship of DpNPV (Diaphania pulverulentalis Nuclear Polyhedrosis Virus) with Other Lepidopteran NPVs based on Polyhedrin Gene

The mulberry leaf-roller, Diaphania pulverulentalis (Hampson) is a major defoliator pest of mulberry in sericultural tracts of Karnataka, India. A nuclear polyhedrosis virus isolated from D. pulverulentalis larvae was analysed via its polyhedrin gene, which was amplified from DpNPV DNA in polymerase chain reaction using degenerate set of primers and the sequence was analysed phylogenetically with other polyhedrin genes. The analysis revealed that DpNPV was closely related to Galleria mellonella NPV, Plutella maculipennis NPV and Autographa californica NPV and Autographa biloba NPV and distinct from Bombyx mori NPV.

Inorganic binding peptide‐mediated immobilization based on baculovirus surface display system

The biomolecule-mediated assembly of novel composites has been the subject of numerous investigations during the last years, providing new insights into material science and engineering. Via molecular biology technology, we were able to introduce the genetically engineered polypeptide for inorganics (GEPI) as a molecular binder into biomolecules such as phage viruses, to assemble hybrid functional nanoarchitectures. In the present work, we introduced a novel nanocomposite comprising the Autographa californica nuclear polyhedrosis virus (AcNPV) and nanoparticles bound to it. Our results show that a GEPI-encoding gene was successfully introduced by recombination into a eukaryotic expression bacmid and finally displayed outside of the AcNPV after transfection into Sf9 insect cells using the Bac-to-Bac baculovirus expression system. The recombinant baculovirus maintained both the viral infectivity and the specific binding activity of the GEPI. The construction of the gene in the recombinant plasmid was examined by polymerase chain reaction analysis and enzymatic digestion identification, and verified by gene sequencing. Surface display of the fused peptide was revealed by Western blot analysis in dissolution studies and determined by immuno- gold electron microscopy. Adherence of nanoparticles to the recombinant baculovirus was visualized by transmission electron microscopy analysis. Here, we demonstrated the possibilities of combining peptide-mediated immobilization with baculovirus surface display technology.

Baculovirus for Eukaryotic Protein Display

The baculovirus Autographa californica nuclear polyhedrosis virus is an attractive candidate for eukaryotic virus display. A variety of strategies exists to incorporate and present target proteins on the surface of infected insect cells as well as on budded virions. Native baculovirus proteins such as the major envelope protein and the capsid protein, but also foreign scaffolds such as the vesicular stomatitis virus G-1 protein or the influenza A virus hemagglutinin serve as fusion partners for surface presentation of target proteins. The purposes of surface display are manifold; efficient presentation of antigenic epitopes on budded virions serves to induce specific immune response, designed surface odifications alter the binding properties and host specificities of the baculovirus to e.g. mammalian cells and help to enhance mammalian cell transduction. Eukaryotic surface display libraries based on the baculovirus system allow selecting for specific binding proteins while providing post translational modifications. Here we describe the different possibilities and strategies to accessibly present foreign proteins and peptides on the surface of insect cells and baculoviruses, as well as the applications thereof, including vector design, cloning strategies and construction and screening of surface expression libraries.

Host Innate Immune Responses Induced by Baculovirus in Mammals

The baculovirus Autographa californica nuclear polyhedrosis virus has been widely used not only to achieve a high level of foreign gene expression in insect cells but also for efficient gene transduction into mammalian cells without any replication. In addition to the efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. The baculovirus has abundant CpG motifs in the viral genome and is capable of inducing pro-inflammatory cytokines and interferons through Toll-like receptor-dependent and -independent signaling pathways in a cell-type-specific manner. The baculovirus also has a strong adjuvant activity, and recombinant baculoviruses encoding neutralization epitopes elicit protective immunity in mice. This review deals with the current status of our knowledge of the induction of host innate immune responses by baculovirus and discusses the future prospects for baculovirus vectors.

Baculovirus treatment fully protects mice against a lethal challenge of FMDV

Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease that affects cattle, swine, goat and sheep among others. FMDV is able to overcome the initial host innate immune response by inhibiting the induction of antiviral molecules at both the transcriptional and the translational levels. It has been demonstrated that FMDV A/Arg/2001 causes the death of adult C57Bl/6 mice within 72h. We evaluated the capacity of Autographa californica nuclear polyhedrosis virus (AcNPV), an insect virus with potent innate immunostimulating effects, to promote early protection against FMDV A/Arg/2001 challenge in C57Bl/6 mice. Groups of 8–9 weeks old female mice were injected intravenously with AcNPV and challenged with a lethal dose of FMDV at different times post-administration. Our results showed that pretreatment of mice with a single injection of AcNPV 3h or 3 days before FMDV challenge resulted in complete abrogation of mortality and complete or partial suppression of viremia, respectively. Furthermore, no signs of disease were observed. AcNPV could be a valuable tool to improve the design of a novel vaccine that protects as an adjuvant at early times post-vaccination.

Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.