Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1

IntroductionBaculoviruses are enveloped viruses that have doublestranded, circular DNA genomes ranging in size from 80 to 180 kbp (1,2). Baculoviruses have been isolated from more than 600 insect species belonging to the orders of Lepidoptera, Hymenoptera, Diptera, Orthoptera, Coleoptera, Neuroptera, Thysanura, and Trichoptera (3). The most notable characteristic of baculoviruses is the occlusion body (OB). The occlusion body is a crystalline matrix composed of a protein called polyhedrin, which provides protection to the virions in the environment against proteolytic and chitinolytic enzymes in the decomposing larvae and spreads infection among insects (4,5). Baculoviruses have been taxonomically divided based on their OB morphology into nucleopolyhedroviruses (NPVs) and granuloviruses (GVs), forming polyhedra and granula, respectively (6). In the NPV, the OB ranges in size from 0.4 to 5 µm in diameter and contains several virions (7,8). The OBs of NPVs are most easily seen under light microscope due to their larger size and their light refractory polyhedral structure. The OBs of GVs, called granula, appear as dark granules and are comparatively more difficult to resolve under light microscope. They are ovoid-shaped and about 500 nm long and 20 nm wide (8,9).The family Baculoviridae is divided into 4 genera according to common biological and structural characteristics and patterns of host associations: Alphabaculovirus, which includes lepidopteran-specific baculoviruses and is subdivided into Group I or Group II based on the phylogenetic analysis of the polyhedrin genes from different baculoviruses; Betabaculovirus, comprising lepidopteran-specific granuloviruses; Gammabaculovirus, which includes hymenopteran-specific baculoviruses; and finally Deltabaculovirus, which contains dipteran-specific baculoviruses (4,10,11).Malacosoma neustria (Linnaeus, 1758) (Lepidoptera: Lasiocampidae), known as the European tent caterpillar, is an important defoliator of various fruit trees such as apple, pear, and plum; wild and ornamental trees and shrubs, including oak and rose species, oleaster, sea buckthorn, barberry, elm trees, willow, poplar, and aspen; and birch, particularly in eastern and central Turkey (12)(13)(14). The caterpillars first eat the buds and then leaves of the trees. During some years their population reaches such high numbers that they leave the trees completely leafless. Factors such as weather conditions and natural enemies including predators, parasitoids, and pathogenic microorganisms have historically been important regulatory elements in the population cycles of this insect.The key microbial pathogens of this insect include NPVs (15). The presence of NPVs in M. neustria was reported by several authors (16-31). In 2009, Demir et al.

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“…Viral DNA was extracted from the polyhedra-derived virus particles according to the method described by Reed et al (35) …”

Section: Isolation Of Viral Dna and Restriction Enzyme Analysismentioning

confidence: 99%

Characterization of a new isolate of Malacosoma neustria nucleopolyhedrovirus (ManeNPV) from Turkey

Demir

Nalçacıoğlu²,

Gholizad³

et al. 2013

Turk J Biol

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“…Virions were released from OBs by incubation in 0.1 M Na 2 CO 3 for 15 min at 37 uC. DNA was extracted according to the method described by Reed et al (2003). After phenol/chloroform extraction, DNA was dialysed for 48 h against 1 mM Tris/HCl, 0.1 mM EDTA (pH 8.0).…”

Section: Methodsmentioning

confidence: 99%

Host-range expansion of Spodoptera exigua multiple nucleopolyhedrovirus to Agrotis segetum larvae when the midgut is bypassed

Jakubowska

Lynn

Herrero

et al. 2009

Journal of General Virology

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Given the high similarity in genome content and organization between Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and Agrotis segetum nucleopolyhedrovirus (AgseNPV), as well as the high percentages of similarity found between their 30 core genes, the specificity of these NPVs was analysed for the respective insect hosts, S. exigua and A. segetum. The LD 50 for AgseNPV in second-instar A. segetum larvae was 83 occlusion bodies per larva and the LT 50 was 8.1 days. AgseNPV was orally infectious for S. exigua, but the LD 50 was 10 000-fold higher than for SeMNPV. SeMNPV was not infectious for A. segetum larvae when administered orally, but an infection was established by injection into the haemocoel. Bypassing midgut entry by intrahaemocoelic inoculation suggested that the midgut is the major barrier in A. segetum larvae for infection by SeMNPV. Delayed-early genes of SeMNPV are expressed in the midgut of A. segetum larvae after oral infections, indicating that the virus is able to enter midgut epithelial cells and that it proceeds through the first phases of the infection process. The possible mechanisms of A. segetum resistance to SeMNPV in per os infections are discussed.

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“…DNA was isolated according to Reed et al (2003) with the modiWcation of using dialysis following phenol:chloroform:isoamyl alcohol extraction. DNA solution was dialyzed against three changes of TE buVer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) at 4°C for 24-48 h. For restriction enzyme analyses 1 g of DNA was digested for 3.5 h at 37°C with HindIII, NotI or PstI, electrophoresed in 0.7% TAE [40 mM Tris-acetate, 1 mM EDTA (pH 8.0)], separated in 0.6% agarose gels at 15 mA for 18 h and analyzed under UV light after staining the DNA with ethidium bromide.…”

Section: Dna Extraction and Restriction Enzyme Analysismentioning

confidence: 99%

European Leucoma salicis NPV is closely related to North American Orgyia pseudotsugata MNPV

Jakubowska¹,

Oers²,

Cory³

et al. 2005

Journal of Invertebrate Pathology

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Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1

Cited by 21 publications

References 16 publications

Characterization of a new isolate of Malacosoma neustria nucleopolyhedrovirus (ManeNPV) from Turkey

Characterization of a new isolate of Malacosoma neustria nucleopolyhedrovirus (ManeNPV) from Turkey

Host-range expansion of Spodoptera exigua multiple nucleopolyhedrovirus to Agrotis segetum larvae when the midgut is bypassed

European Leucoma salicis NPV is closely related to North American Orgyia pseudotsugata MNPV

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