Comparative genomics of the Neodiprion sertifer nucleopolyhedrovirus from Turkey with the fewest ORFs among baculoviruses.

The complete genome of a European pine sawfly Neodiprion sertifer nucleopolyhedrovirus (NeseNPV-TR) was sequenced and characterized from next-generation sequencing data of N. sertifer larva from Türkiye. This genome was analyzed and compared to previously reported genomes of baculoviruses. The baculovirus phylogeny was reconstructed and the species identity of the NeseNPV-TR was delineated using K2P distance. The length of the genome was 82,052 bp, with a G + C content of 33.28%. It contained 83 putative ORFs, including 38 baculovirus core genes, three lepidopteran baculovirus core genes, and three non-conserved genes. It had five hrs with 20.6% overall mean distance on average. The pairwise K2P distances of lef-8, lef-9, and polh genes and combinations of three genes and 38 genes between NeseNPV-TR and NeseNPV were slightly higher than the specified threshold values for species demarcation. The most variable genes were lef-2, helicase, p40, desmoplakin, pif7, p6.9, vp91, and vp39, while the most conserved were lef-8, lef-9, odv-e18, pif2, and lef-5 among baculoviruses. The genome of NeseNPV-TR is smaller and contains the fewest ORFs among baculoviruses. Some of unassigned ORFs had conserved domains and hence, we suggest further investigation to determine their structural and functional roles. Phylogenetic analyses confirmed its position within genus Gammabaculovirus. Taking into account the phylogenetic position, K2P distances, and NJ tree, the NeseNPV-TR can be classified in the same species (Gammabaculovirus nesertiferis) with NeseNPV. The different divergence rates in the baculovirus core genes may be related with different selection pressures acting on the genes. The lower genetic diversity of Group I alphabaculoviruses is most probably due to recent emergence.

Transcriptomics and interactomics during the primary infection of an SfNPV baculovirus on Spodoptera frugiperda larvae.

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

Naturally Occurring Yeasts Associated with Thaumatotibia leucotreta Can Enhance the Efficacy of the Cryptophlebia Leucotreta Granulovirus.

Yeasts associated with lepidopteran pests have been shown to play a role in their survival, development, and oviposition preference. It has been demonstrated that combining these yeasts with existing biological control agents can enhance their efficacy. The tortricid Thaumatotibia leucotreta is a phytosanitary pest in the South African citrus industry, with the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV) being one of the components that can control this pest. Several yeast species were shown to be associated with T. leucotreta larvae, which affected their behaviour and development. A series of detached fruit bioassays were performed to determine whether the combination of yeast with CrleGV enhances its efficacy. These assays included determining the optimal yeast/virus ratio, testing all isolated yeast species in combination with CrleGV, and further improving yeast/virus formulation by adding an adjuvant. The optimal yeast concentration to use alongside CrleGV was determined to be 106 cells·mL-1. Pichia kluyveri, P. kudriavzevii, Kluyveromyces marxianus, and Saccharomyces cerevisiae in combination with CrleGV reduced larval survival compared to CrleGV alone. The addition of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae in combination with CrleGV did not notably improve their effectiveness; however, there was an observed decrease in larval survival. In future studies, field trials will be conducted with combinations of CrleGV and P. kudriavzevii or S. cerevisiae to investigate whether these laboratory findings can be replicated in orchard conditions.

The History of Baculovirology in Africa.

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.

Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells.

Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z' value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3R cells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies.

On-farm Production of Microbial Entomopathogens for use in Agriculture: Brazil as a Case Study.

In Brazil, the production of beneficial microorganisms by growers exclusively for their own use is a practice known as "on-farm production". Regarding on-farm bioinsecticides, they were initially deployed for pests of perennial and semi-perennial crops in the 1970s but, since 2013, their use has extended to pests of annual crops such as maize, cotton, and soybean. Millions of hectares are currently being treated with these on-farm preparations. Local production reduces costs, meets local needs, and reduces inputs of environmentally damaging chemical pesticides, facilitating establishment of more sustainable agroecosystems. Critics argue that without implementation of stringent quality control measures there is the risk that the on-farm preparations: (1) are contaminated with microbes which may include human pathogens or (2) contain very little active ingredient, impacting on field efficacy. The on-farm fermentation of bacterial insecticides predominates, especially that of Bacillus thuringiensis targeting lepidopteran pests. However, there has been a rapid growth in the past 5 years in the production of entomopathogenic fungi, mostly for the control of sap-sucking insects such as whitefly (Bemisia tabaci (Gennadius)) and the corn leafhopper (Dalbulus maidis (DeLong and Wolcott)). In contrast, on-farm production of insect viruses has seen limited growth. Most of the ca. 5 million rural producers in Brazil own small or medium size properties and, although the vast majority still do not practice on-farm production of biopesticides, the topic has aroused interest among them. Many growers who adopt this practice usually use non-sterile containers as fermenters, resulting in poor-quality preparations, and cases of failure have been reported. On the other hand, some informal reports suggest on-farm preparations may be efficacious even when contaminated, what could be explained, at least partially, by the insecticidal secondary metabolites secreted by the pool of microorganisms in the liquid culture media. Indeed, there is insufficient information on efficacy and mode of action of these microbial biopesticides. It is usually the large farms, some with > 20,000 ha of continuous cultivated lands, that produce biopesticides with low levels of contamination, as many of them possess advanced production facilities and have access to specialized knowledge and trained staff. Uptake of on-farm biopesticides is expected to continue but the rate of adoption will depend on factors such as the selection of safe, virulent microbial strains and implementation of sound quality control measures (compliance with emerging Brazilian regulations and international standards). The challenges and opportunities of on-farm bioinsecticides are presented and discussed.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

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

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.

The naked truth: An updated review on nudiviruses and their relationship to bracoviruses and baculoviruses.

Nudiviruses (Nudiviridae) are double-stranded DNA viruses with enveloped and rod-shaped virions. Several insect orders (e.g., Diptera, Lepidoptera, Coleoptera, Orthoptera) and aquatic crustaceans are susceptible to nudivirus infections, which can result in varied degrees of disease in all developmental host stages. Their pathogenicity endangers insect rearing and crustacean aquacultures, but has also proven effective in biocontrol against Oryctes rhinoceros infestations. This literature review aims to present all known nudivirus species and provide a comprehensive Nudiviridae phylogeny by including recently described nudiviral isolates, and discuss this phylogeny in comparison to current opinions and taxonomical propositions. Moreover, we aim to clarify biological, pathological and genomic differences or similarities between nudiviruses and related entomopathogenic viruses, including baculoviruses (Baculoviridae) and bracoviruses (Polydnaviridae). A phylogenetic analysis using 17 concatenated nudivirus core genes resulted in the expected structure with the genera Alphanudivirus and Betanudivirus, as well as the most recently recognized genera Gammanudivirus and Deltanudivirus. The hymenopteran Osmia cornuta nudivirus (OcNV) groups closest with the hymenopteran Fopius arisanus endogenous nudivirus (FaENV) and does not share a most common ancestor with the hymenopteran bracoviruses. Except for one node, all clades are highly supported. The proposition of a recent study to assign subgroups to the alphanudiviruses might be legitimate, but more hymenopteran and orthopteran nudiviruses, especially in bees and cricket, need to be identified to resolve this proposal. In addition, freshwater and marine nudiviruses might form taxonomic subgroups among gammanudiviruses as well, but more aquatic nudiviruses need to be identified and sequenced for better resolution. Furthermore, the search for nudiviruses in insects with (semi)aquatic life stages may aid in finding the missing link that led to the manifestation of aquatic nudiviruses.

CpGV-M Replication in Type I Resistant Insects: Helper Virus and Order of Ingestion Are Important.

The genetic diversity of baculoviruses provides a sustainable agronomic solution when resistance to biopesticides seems to be on the rise. This genetic diversity promotes insect infection by several genotypes (i.e., multiple infections) that are more likely to kill the host. However, the mechanism and regulation of these virus interactions are still poorly understood. In this article, we focused on baculoviruses infecting the codling moth, Cydia pomonella: two Cydia pomonella granulovirus genotypes, CpGV-M and CpGV-R5, and Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV). The influence of the order of ingestion of the virus genotypes, the existence of an ingestion delay between the genotypes and the specificity of each genotype involved in the success of multiple infection were studied in the case of Cydia pomonella resistance. To obtain a multiple infection in resistant insects, the order of ingestion is a key factor, but the delay for ingestion of the second virus is not. CrpeNPV cannot substitute CpGV-R5 to allow replication of CpGV-M.

Western equine encephalitis virus virus-like particles from an insect cell-baculovirus system elicit the strong immune responses in mice.

Western equine encephalitis virus (WEEV) causes lethal encephalitis in humans and equines, and it poses a serious public health threat in many countries. Therefore, the development of an efficient vaccine remains an important challenge for the prevention of WEEV infection. This study presents the first description of WEEV virus-like particles (VLPs) generated from insect cells using recombinant baculoviruses. WEEV VLPs with 206 adjuvant could trigger a strong cellular immune response; increase the levels of IL-2, IL-4 and IFN-γ; and induce a high level of neutralizing antibodies against WEEV in mice. These data showed that the insect cell-baculovirus system is suitable for the production of WEEV VLPs and that these VLPs could elicit the strong immunogenicity in mice. These results suggest a new, nonreplicating, and effective vaccine candidate against WEEV infection.

Global Metabolic Profiling of Baculovirus Infection in Silkworm Hemolymph Shows the Importance of Amino-Acid Metabolism.

Viruses rely on host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Infection of the silkworm, Bombyx mori, by Bombyx mori nucleopolyhedrovirus (BmNPV), has been studied extensively in the past to unravel interactions between baculoviruses and their lepidopteran hosts. To understand the interaction between the host metabolic responses and BmNPV infection, we analyzed global metabolic changes associated with BmNPV infection in silkworm hemolymph. Our metabolic profiling data suggests that amino acid metabolism is strikingly altered during a time course of BmNPV infection. Amino acid consumption is increased during BmNPV infection at 24 h post infection (hpi), but their abundance recovered at 72 hpi. Central carbon metabolism, on the other hand, particularly glycolysis and glutaminolysis, did not show obvious changes during BmNPV infection. Pharmacologically inhibiting the glycolytic pathway and glutaminolysis also failed to reduce BmNPV replication, revealing that glycolysis and glutaminolysis are not essential during BmNPV infection. This study reveals a unique amino acid utilization process that is implemented during BmNPV infection. Our metabolomic analysis of BmNPV-infected silkworm provides insights as to how baculoviruses induce alterations in host metabolism during systemic infection.

Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC4 Receptors in Budded Baculoviruses.

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC4) receptor.

Dataset on recombinant expression of an ancient chitinase gene from different species of Leishmania parasites in bacteria and in Spodoptera frugiperda cells using baculovirus.

The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.

Entomopathogenic Viruses in the Neotropics: Current Status and Recently Discovered Species.

The market for biological control of insect pests in the world and in Brazil has grown in recent years due to the unwanted ecological and human health impacts of chemical insecticides. Therefore, research on biological control agents for pest management has also increased. For instance, insect viruses have been used to protect crops and forests around the world for decades. Among insect viruses, the baculoviruses are the most studied and used viral biocontrol agent. More than 700 species of insects have been found to be naturally infected by baculoviruses, with 90% isolated from lepidopteran insects. In this review, some basic aspects of baculovirus infection in vivo and in vitro infection, gene content, viral replication will be discussed. Furthermore, we provide examples of the use of insect viruses for biological pest control and recently characterized baculoviruses in Brazil.

Evaluating the Immune Response of Recombinant H1N1 Hemagglutinin with MF59 Adjuvant in Animal Model as a Novel Alternative to the Influenza Vaccine.

The H1N1 influenza virus is known as a serious pandemic threat across the globe. Vaccination is one of the most effective methods of protection against this virus and the way to reduce the seasonal pandemic risk. The commercial vaccine does not adequately respond to pandemic strains. This study examines the potential function of formulated H1N1 hemagglutinin with MF59 adjuvant against A/PR/8/34 (H1N1). To this end, a recombinant hemagglutinin (rHA) gene of influenza A virus was designed and expressed in SF9 cell by the Baculovirus expression system. Four groups of mice were immunized by rHA in combination with MF59, Alum adjuvant, and virus split only. The immunized mice subsequently used for the humoral immune assay and the results compared with untreated mice (negative group). Besides, both treated and control mice groups were challenged with mouse-adapted influenza virus A/PR/8/34(H1N1) through the intranasal drop. Bodyweight, survival, temperature variation, and the medical conditions of the samples were assessed. Mice immunized with the recombinant protein demonstrated a humoral response to the influenza A virus. Upon virus challenging, co-administration of rHA with MF59 adjuvant could lead to 92% survival of the vaccinated mice within 10 days. The MF59-treated group showed slight weight loss and high-temperature body two weeks after infection. This group also displayed a higher hemagglutination inhibition (HI) antibody titer as compared to the group vaccinated with virus split, and Alum adjuvant. Altogether, the results showed that the recombinant protein with the MF59 adjuvant created better safety than the Alum adjuvant, thereby can be considered as a safe and reliable vaccine against the H1N1 virus for further investigations.

How far have we reached in development of effective influenza vaccine?

Despite of ongoing research programs and numerous clinical trials, seasonal influenza epidemics remain a major concern globally. Vaccination remains the most effective method to prevent influenza infection. However, current flu vaccines have several limitations, including limited vaccine capacity, long production times, inconsistence efficacy in certain populations, and lack of a "universal" solution. Different next-generation approaches such as cell line-based culture, reverse genetics, and virus expression technology are currently under development to address the aforementioned challenges in conventional vaccine manufacture pipeline. Such approaches hope for safe and scalable production, induce broad-spectrum immunity, create premade libraries of vaccine strains, and target nonvariable regions of antigenic proteins for "universal" vaccination. Here, we discuss the process and challenges of the current influenza vaccine platform as well as new approaches that are being investigated. These developments indicate that an exciting future lies ahead in the influenza vaccine field.

Kinesin-1 Proteins KIF5A, -5B, and -5C Promote Anterograde Transport of Herpes Simplex Virus Enveloped Virions in Axons.

Herpes simplex virus (HSV) and other alphaherpesviruses must spread from sites of viral latency in sensory ganglia to peripheral tissues, where the viruses can replicate to higher titers before spreading to other hosts. These viruses move in neuronal axons from ganglia to the periphery propelled by kinesin motors moving along microtubules. Two forms of HSV particles undergo this anterograde transport in axons: (i) unenveloped capsids that become enveloped after reaching axon tips and (ii) enveloped virions that are transported within membrane vesicles in axons. Fundamental to understanding this axonal transport is the question of which of many different axonal kinesins convey HSV particles. Knowing which kinesins promote axonal transport would provide clues to the identity of HSV proteins that tether onto kinesins. Prominent among axonal kinesins are the kinesin-1 (KIF5A, -5B, and -5C) and kinesin-3 (e.g., KIF1A and -1B) families. We characterized fluorescent forms of cellular cargo molecules to determine if enveloped HSV particles were present in the vesicles containing these cargos. Kinesin-1 cargo proteins were present in vesicles containing HSV particles, but not kinesin-3 cargos. Fluorescent kinesin-1 protein KIF5C extensively colocalized with HSV particles, while fluorescent kinesin-1 KIF1A did not. Silencing of kinesin-1 proteins KIF5A, -5B, and -5C or light chains KLC1 and KLC2 inhibited the majority of HSV anterograde transport, while silencing of KIF1A had little effect on HSV transport in axons. We concluded that kinesin-1 proteins are important in the anterograde transport of the majority of HSV enveloped virions in neuronal axons and kinesin-3 proteins are less important.IMPORTANCE Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether onto dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde) or from cell bodies to axon tips (anterograde). Following reactivation from latency, alphaherpesviruses absolutely depend upon anterograde transport of virus particles in axons in order to reinfect peripheral tissues and spread to other hosts. Which of the many axonal kinesins transport HSV in axons is not clear. We characterized fluorescent cellular cargo molecules and kinesins to provide evidence that HSV enveloped particles are ferried by kinesin-1 proteins KIF5A, -5B, and -5C and their light chains, KLC1 and KLC2, in axons. Moreover, we obtained evidence that kinesin-1 proteins are functionally important in anterograde transport of HSV virions by silencing these proteins.

Assays with Detection of Fluorescence Anisotropy: Challenges and Possibilities for Characterizing Ligand Binding to GPCRs.

Binding of fluorescent ligands (tracers) to their target receptors can be directly monitored over time, as the binding of a low-molecular-weight (LMW) tracer to a larger particle causes an increase of fluorescence anisotropy (FA). The combination of bright fluorophores, tracers with low nonspecific binding, and budded baculovirus particles (BVPs) for overexpression of G protein-coupled receptors (GPCRs) ensures a high signal-to-noise ratio in FA assays. The obtained data enable quantitative assessment of equilibrium binding and kinetic parameters for both the tracer and competing compounds as well as an estimation of the receptor concentration. FA assays have clear potential for implementation in drug screening systems, but also in studies of ligand-binding mechanisms for particular GPCRs.

Agrotis segetum nucleopolyhedrovirus but not Agrotis segetum granulovirus replicate in AiE1611T cell line of Agrotisipsilon.

Both Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV) belong to a cluster of four baculoviruses that are infective for different Agrotis species. Belonging further to different baculovirus genera, namely Alphabaculovirus and Betabaculovirus, respectively, AgseNPV-B and AgseGV are candidates to investigate virus interactions in co-infections. However, for the investigation of virus interactions on a cellular level, permissive insect cell-lines are needed. The cell line AiE1611T deriving from Agrotisipsilon eggs has been shown to be permissive for several Alphabaculovirus isolates. In this study, virus replication was followed based on microscopic analysis of infected and transfected cells, as well as on a molecular level by PCR of DNA and cDNA of selected baculovirus transcripts. While the permissivity was not verified for AgseGV, AgseNPV-B produced occlusion bodies in both infection with hemolymph of infected larvae and Lipofectamin transfection with AgseNPV-B genomic DNA. In addition to the possibility to investigate virus interaction of AgseNPV-B with other alphabaculoviruses, the permissivity of AiE1611T for AgseNPV-B further offers the possibility a biological selection to separate AgseNPV-B from AgseGV.