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      Comprehensive analysis of single molecule sequencing-derived complete genome and whole transcriptome of Hyposidra talaca nuclear polyhedrosis virus

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          Abstract

          We sequenced the Hyposidra talaca NPV (HytaNPV) double stranded circular DNA genome using PacBio single molecule sequencing technology. We found that the HytaNPV genome is 139,089 bp long with a GC content of 39.6%. It encodes 141 open reading frames (ORFs) including the 37 baculovirus core genes, 25 genes conserved among lepidopteran baculoviruses, 72 genes known in baculovirus, and 7 genes unique to the HytaNPV genome. It is a group II alphabaculovirus that codes for the F protein and lacks the gp64 gene found in group I alphabaculovirus viruses. Using RNA-seq, we confirmed the expression of the ORFs identified in the HytaNPV genome. Phylogenetic analysis showed HytaNPV to be closest to BusuNPV, SujuNPV and EcobNPV that infect other tea pests, Buzura suppressaria, Sucra jujuba, and Ectropis oblique, respectively. We identified repeat elements and a conserved non-coding baculovirus element in the genome. Analysis of the putative promoter sequences identified motif consistent with the temporal expression of the genes observed in the RNA-seq data.

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          Insect pathogens as biological control agents: Back to the future.

          The development and use of entomopathogens as classical, conservation and augmentative biological control agents have included a number of successes and some setbacks in the past 1years. In this forum paper we present current information on development, use and future directions of insect-specific viruses, bacteria, fungi and nematodes as components of integrated pest management strategies for control of arthropod pests of crops, forests, urban habitats, and insects of medical and veterinary importance. Insect pathogenic viruses are a fruitful source of microbial control agents (MCAs), particularly for the control of lepidopteran pests. Most research is focused on the baculoviruses, important pathogens of some globally important pests for which control has become difficult due to either pesticide resistance or pressure to reduce pesticide residues. Baculoviruses are accepted as safe, readily mass produced, highly pathogenic and easily formulated and applied control agents. New baculovirus products are appearing in many countries and gaining an increased market share. However, the absence of a practical in vitro mass production system, generally higher production costs, limited post application persistence, slow rate of kill and high host specificity currently contribute to restricted use in pest control. Overcoming these limitations are key research areas for which progress could open up use of insect viruses to much larger markets. A small number of entomopathogenic bacteria have been commercially developed for control of insect pests. These include several Bacillus thuringiensis sub-species, Lysinibacillus (Bacillus) sphaericus, Paenibacillus spp. and Serratia entomophila. B. thuringiensis sub-species kurstaki is the most widely used for control of pest insects of crops and forests, and B. thuringiensis sub-species israelensis and L. sphaericus are the primary pathogens used for control of medically important pests including dipteran vectors. These pathogens combine the advantages of chemical pesticides and MCAs: they are fast acting, easy to produce at a relatively low cost, easy to formulate, have a long shelf life and allow delivery using conventional application equipment and systemics (i.e. in transgenic plants). Unlike broad spectrum chemical pesticides, B. thuringiensis toxins are selective and negative environmental impact is very limited. Of the several commercially produced MCAs, B. thuringiensis (Bt) has more than 50% of market share. Extensive research, particularly on the molecular mode of action of Bt toxins, has been conducted over the past two decades. The Bt genes used in insect-resistant transgenic crops belong to the Cry and vegetative insecticidal protein families of toxins. Bt has been highly efficacious in pest management of corn and cotton, drastically reducing the amount of broad spectrum chemical insecticides used while being safe for consumers and non-target organisms. Despite successes, the adoption of Bt crops has not been without controversy. Although there is a lack of scientific evidence regarding their detrimental effects, this controversy has created the widespread perception in some quarters that Bt crops are dangerous for the environment. In addition to discovery of more efficacious isolates and toxins, an increase in the use of Bt products and transgenes will rely on innovations in formulation, better delivery systems and ultimately, wider public acceptance of transgenic plants expressing insect-specific Bt toxins. Fungi are ubiquitous natural entomopathogens that often cause epizootics in host insects and possess many desirable traits that favor their development as MCAs. Presently, commercialized microbial pesticides based on entomopathogenic fungi largely occupy niche markets. A variety of molecular tools and technologies have recently allowed reclassification of numerous species based on phylogeny, as well as matching anamorphs (asexual forms) and teleomorphs (sexual forms) of several entomopathogenic taxa in the Phylum Ascomycota. Although these fungi have been traditionally regarded exclusively as pathogens of arthropods, recent studies have demonstrated that they occupy a great diversity of ecological niches. Entomopathogenic fungi are now known to be plant endophytes, plant disease antagonists, rhizosphere colonizers, and plant growth promoters. These newly understood attributes provide possibilities to use fungi in multiple roles. In addition to arthropod pest control, some fungal species could simultaneously suppress plant pathogens and plant parasitic nematodes as well as promote plant growth. A greater understanding of fungal ecology is needed to define their roles in nature and evaluate their limitations in biological control. More efficient mass production, formulation and delivery systems must be devised to supply an ever increasing market. More testing under field conditions is required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly, greater attention must be paid to their use within integrated pest management programs; in particular, strategies that incorporate fungi in combination with arthropod predators and parasitoids need to be defined to ensure compatibility and maximize efficacy. Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are potent MCAs. Substantial progress in research and application of EPNs has been made in the past decade. The number of target pests shown to be susceptible to EPNs has continued to increase. Advancements in this regard primarily have been made in soil habitats where EPNs are shielded from environmental extremes, but progress has also been made in use of nematodes in above-ground habitats owing to the development of improved protective formulations. Progress has also resulted from advancements in nematode production technology using both in vivo and in vitro systems; novel application methods such as distribution of infected host cadavers; and nematode strain improvement via enhancement and stabilization of beneficial traits. Innovative research has also yielded insights into the fundamentals of EPN biology including major advances in genomics, nematode-bacterial symbiont interactions, ecological relationships, and foraging behavior. Additional research is needed to leverage these basic findings toward direct improvements in microbial control.
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            A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase.

            The predicted amino acid sequence of a newly identified gene of the insect baculovirus Autographa californica nuclear polyhedrosis virus was similar to several uridine 5'-diphosphate (UDP)-glucuronosyl transferases and at least one UDP-glucosyl transferase. Genetic and biochemical studies confirmed that this gene encodes an ecdysteroid UDP-glucosyl transferase (egt). This enzyme catalyzes the transfer of glucose from UDP-glucose to ecdysteroids, which are insect molting hormones. Expression of the egt gene allowed the virus to interfere with normal insect development so that molting was blocked in infected larvae of fall armyworm (Spodoptera frugiperda).
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              Baculovirus diversity and molecular biology.

              With the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. Since less than 30% of the AcMNPV genome has been sequenced and characterized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our understanding of the baculovirus genome and its replication. The size and diversity of baculovirus genomes appears to be strongly influenced by mobile DNA from the insect host. Also, transposon-mediated mutations of baculoviruses provide examples of functional inactivation of viral genes (FP phenotype mutations) and transcriptional activation (TE-D insertion). Another role transposable elements may play is the introduction of insect promoters and enhancers to the baculovirus genome. Since early baculovirus genes are likely transcribed in a way similar to normal insect genes, transposons that insert strong constitutive promoters or cellular enhancers near early baculovirus genes may cause mutations that are subsequently selected for. If this does occur, baculovirus early gene promoters may exhibit a great deal of variability in sequence and may resemble host promoters. Given the overall similarity between the genomes of OpMNPV and AcMNPV and the apparent absence of a region, similar to the AcMNPV HindIII-K/EcoR1-S in OpMNPV, it is intriguing to speculate that this region which contains two ORFs and the hr5 enhancer, may have been inserted into the AcMNPV genome by transposition, possibly delivering several helpful genes (35k and 94k) and a powerful enhancer. The highly repeated enhancer may have been subsequently amplified by recombination. In such a model, the acquisition of general or species-specific enhancers might influence both virulence and host range. Acquisition of general enhancers could increase the level of early gene expression, thus accelerating the cellular infection cycle and making the virus more virulent. Similarly, the acquisition of species-specific enhancers might affect host range by accelerating the infection cycle, but only in a specific host or cell type. One might therefore postulate that diversity in baculoviruses may reflect not only different selection pressures but also the diversity of mobile DNA within host insect species. Although our understanding of baculovirus diversity and molecular biology is rapidly advancing, many of the fundamental characteristics that define the unique nature of baculoviruses remain poorly understood. One fundamental feature of baculoviruses is the production of the two virion phenotypes, PDV and BV.(ABSTRACT TRUNCATED AT 400 WORDS)
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                Author and article information

                Contributors
                sekar@gene.com
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                12 June 2018
                12 June 2018
                2018
                : 8
                : 8924
                Affiliations
                [1 ]ISNI 0000 0004 0534 4718, GRID grid.418158.1, Department of Molecular Biology, Genentech Inc., ; 1 DNA WAY, South San Francisco, CA 94080 USA
                [2 ]AgriGenome Labs Private Limited, 501, SCK01 Building, Smart City Kochi, Infopark Road, Kakkanad, Kochi, 682 030 India
                [3 ]SciGenom Research Foundation, 3rd Floor, Narayana Health City, #258/A, Bommasandra, Hosur Road, Bangalore, 560 099 India
                [4 ]ISNI 0000 0004 0534 4718, GRID grid.418158.1, Department of Pathology, Genentech Inc, ; 1 DNA WAY, South San Francisco, CA 94080 USA
                [5 ]ISNI 0000 0001 0708 3863, GRID grid.482359.1, Tea Research Association, North Bengal Regional R & D Centre, Nagrakata, ; Jalpaiguri, West Bengal 735 225 India
                [6 ]GRID grid.423340.2, Pacific Biosciences, 1305O’Brien Dr, ; Menlo Park, CA 94025 USA
                [7 ]GRID grid.452841.e, SciGenom Labs Pvt Ltd, ; Plot no: 43A,SDF, 3rd floor, A Block, CSEZ, Kakkanad, Cochin, Kerala 682 037 India
                Author information
                http://orcid.org/0000-0002-6614-7182
                http://orcid.org/0000-0003-4394-2455
                http://orcid.org/0000-0003-3047-4250
                http://orcid.org/0000-0003-4272-6443
                Article
                27084
                10.1038/s41598-018-27084-y
                5997678
                29895987
                1b81b096-1c1a-4300-88f6-f8676563e5e4
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 26 February 2018
                : 25 May 2018
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