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Background Chitin, a long-chain polymer of N-acetylglucosamine, is a major structural component of the insect exoskeleton and the peritrophic membrane (PM). Chitinases are able to effectively break down glycosidic bonds of chitin polymer thus can be used in agriculture to control plant pathogen insects. These enzymes can be synthesized by higher plants, animals, protista, bacteria, and viruses. Results In this study, viral and bacterial chitinases were compared for their potential activity on a laboratory test insect. The genes encoding chitinases of Autographa californica nucleopolyhedrovirus (AcNPV) and Cydia pomonella granulovirus (CpGV) were amplified from genomic DNAs by PCR and cloned into the pET-28a (+) expression vector. The chitinase proteins of these 2 viruses (AcNPV-Chi, CpGV-Chi) and Serratia marcescens chitinase C (ChiC) protein which was previously cloned were overexpressed in Escherichia coli. Expressed proteins were purified and confirmed by western blot analysis as 50, 63, and 68 kDa for AcNPV, CpGV, and S. marcescens chitinases, respectively. Enzyme activities of the chitinases were confirmed. Chitinases were also compared to each other in silico. The insecticidal effects of these proteins were evaluated on Galleria mellonella L. larvae. Bioassays were performed on the 3rd instar larvae for each chitinase protein in triplicate. The results showed that although there were differences in enzymatic activities and domain organizations, all 3 microbial chitinases produced almost the same level of insecticidal activity on the test insect. LC50 and LT50 values were compatible with the mortality results. These results were a preanalysis for comparing the effects of microbial chitinases. Conclusion Potential activity experiments should be carried out on more insects to provide detailed information on the insecticidal effects of bacterial and viral chitinases.

Omurgasız iridesan virüs 6 (IIV6), çeşitli zararlı böcek türlerini düşük oranda enfekte edebilir. Rekombinant DNA teknolojisi ile oluşturulan yeni özelliklere sahip virüsler biyolojik kontrol ajanları olarak etkin bir şekilde kullanılabilir. Daha önce IIV6 157L ORF’si yerine birisi yeşil floresan protein geni (rCIV-Δ157L-gfp) ve diğeri de gfp ile birleştirilmiş bir akrep Androctonus australis (Linnaeus, 1758) böcek toksin geni (rCIV-Δ157L/gfp-AaIT) ihtiva eden iki rekombinant IIV6 inşa edilmişti. Bu çalışmada, yaban tip IIV6 ve iki rekombinant virüs, Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae), Spodoptera littoralis (Boisduval, 1883) (Lepidoptera: Noctuidae), Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae), Euproctis chrysorrhoea (Linnaeus, 1758) (Lepidoptera: Erebidae) ve Tenebrio molitor (Linnaeus, 1758) (Coleoptera: ‎Tenebrionidae) larvalarında enfeksiyon oluşturma yeteneği açısından değerlendirildi. Çalışma 2018 ve 2019 yılları arasında Karadeniz Teknik Üniversitesi Biyoloji Bölümü'nde gerçekleştirildi. Her bir böcek larvasını enjekte etmek için virüslerin beş farklı konsantrasyonu (103, 104, 105, 106 ve107 TCID50/ml) kullanıldı. rCIV-Δ157L/gfp-AaIT ile enfekte olmuş S. littoralis dışında tüm larvalar felç oldu. rCIV-Δ157L/gfp-AaIT ile enfekte olmuş böcek larvalarının LC50'si sırasıyla, S. littoralis, T. molitor, L. dispar, H. armigera ve E. chrysorrhoea üzerinde 0.3 x 107, 0.7 x 105, 0.2 x 105, 0.15 x 105, 0.7 x 104 TCID50/ml olarak belirlenmiştir. En yüksek virüs konsantrasyonlarına göre hesaplanan LT50 değerleri, rCIV-Δ157L/gfp-AaIT için S. littoralis, T. molitor, L. dispar, H. armigera ve E. chrysorrhoea'da sırasıyla 10.5, 6.2, 4.7, 7.5 ve 5 gün olarak bulundu. Bu çalışma, rekombinant IIV6'nın Lepidoptera ve Coleoptera takımlarına ait bazı böceklerde patojeniteyi artırdığını göstermiştir., Invertebrate iridescent virus 6 (IIV6) can infect a broad range of pest insect species. Viruses with new features created by recombinant DNA technology can be used effectively as biological control agents. Previously, recombinants have been constructed: IIVs harboring green fluorescent protein gene (gfp) in place of IIV6 157L ORF (rCIV-Δ157L-gfp) and a scorpion Androctonus australis (Linnaeus, 1758) insect toxin gene (AaIT) fused with gfp (rCIV-Δ157L/gfp-AaIT). In this study, wild type IIV6 and the two recombinants, were evaluated for their ability to cause infections on Helicoverpa armigera (Hübner, 1805) (Lepidoptera: Noctuidae), Spodoptera littoralis (Boisduval, 1883) (Lepidoptera: Noctuidae), Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae), Euproctis chrysorrhoea (Linnaeus, 1758) (Lepidoptera: Erebidae) and Tenebrio molitor (Linnaeus, 1758) (Coleoptera: ‎Tenebrionidae) larvae. This study was performed at Karadeniz Technical University, Department of Biology during 2018 and 2019. Five different concentrations (103, 104, 105, 106 and 107 TCID50/ml) of viruses were used to inject each insect larvae. All larvae, infected with rCIV-Δ157L/gfp-AaIT, became paralyzed, except S. littoralis. The LC50 of insect larvae infected by rCIV-Δ157L/gfp-AaIT were determined as 0.3 x 107, 0.7 x 105, 0.2 x 105, 0.15 x 105, 0.7 x 104 TCID50/ml on S. littoralis, T. molitor, L. dispar, H. armigera and E. chrysorrhoea, respectively. LT50 values, calculated according to the highest virus concentrations, were found as 10.5, 6.2, 4.7, 7.5 and 5 d on S. littoralis, T. molitor, L. dispar, H. armigera and E. chrysorrhoea, respectively, for rCIV-Δ157L/gfp-AaIT. This study showed that recombinant IIV6 has increased pathogenicity on some insects from Lepidoptera and Coleoptera.

Invertebrate iridescent virus 6 (IIV6) is the type species of the Iridovirus genus in the Betairidovirinae subfamily of the Iridoviridae family. Transcription of the 215 predicted IIV6 genes is temporally regulated, dividing the genes into three kinetic classes: immediate-early (IE), delayed-early (DE), and late (L). So far, the transcriptional class has been determined for a selection of virion protein genes and only for three genes the potential promoter regions have been analyzed in detail. In this study, we investigated the transcriptional class of all IIV6 genes that had not been classified until now. RT-PCR analysis of total RNA isolated from virus-infected insect cells in the presence or absence of protein and DNA synthesis inhibitors, placed 113, 23 and 22 of the newly analyzed viral ORFs into the IE, DE and L gene classes, respectively. Afterwards, in silico analysis was performed to the upstream regions (200 bp) of all viral ORFs using the MEME Suite Software. The AA(A/T)(T/A)TG(A/G)A and (T/A/C)(T/G/C)T(T/A)ATGG motifs were identified in the upstream region of IE and DE genes, respectively. These motifs were validated by luciferase reporter assays as crucial sequences for promoter activity. For the L genes two conserved motifs were identified for all analyzed genes: (T/G)(C/T)(A/C)A(T/G/C)(T/C)T(T/C) and (C/G/T)(G/A/C)(T/A)(T/G) (G/T)(T/C). However, the presence of these two motifs did not influence promoter activity. Conversely, the presence of these two sequences upstream of the reporter decreased its expression. Single nucleotide mutations in the highly conserved nucleotides at the end of the second motif (TTGT) showed that this motif acted as a repressor sequence for late genes in the IIV6 genome. Next, upstream sequences of IIV6 L genes from which we removed this second motif in silico, were re-analyzed for the presence of potential conserved promoter sequences. Two additional motifs were identified in this way for L genes: (T/A)(A/T)(A/T/G)(A/T)(T/C)(A/G)(A/C)(A/C) and (C/G)(T/C)(T/A/C)C(A/T)(A/T)T(T/G) (T/G)(T/G/A). Independent mutations in either motif caused a severe decrease in luciferase expression. Information on temporal classes and upstream regulatory sequences will contribute to our understanding of the transcriptional mechanisms in IIV6.

Previously we have generated a recombinant Chilo iridescent virus (CIV) by inserting the green fluorescent protein gene (gfp) into the CIV 157L open reading frame (ORF) locus and showed that this recombinant (rCIV-Delta 157L-gfp) was fully infectious both in cell culture as well as in insect larvae. This study opened up a new avenue for increasing the speed of kill of CIV and other iridoviruses by inserting virulence or toxin genes into the viral genome. In the current study we constructed a recombinant CIV (rCIV-Delta 157L/gfp-AaIT) where the 157L ORF was replaced with both the AaIT neurotoxin gene from the scorpion Androctonus australis and the gfp gene, each under control of the viral major capsid protein (mcp) gene promoter. Recombinant virus was purified by successive rounds of plaque purification using Spodoptera frugiperda (Sf-9) cells. One-step growth curves for the recombinant viruses, rCIV-Delta 157L/gfp-AaIT and rCIV-Delta 157L-gfp, and wild-type CIVs in Sf-9 cells showed similar profiles. AaIT toxin expression in infected third instar Galleria mellonella larvae was confirmed by western blot analysis using an antibody against the AaIT protein. rCIV-Delta 157L/gfp-AaIT infection at a concentration that kills 100% of the larvae caused paralysis in infected third instar G. mellonella larvae from two days after injection, whereas infection with non-AaIT containing viruses showed mortality starting much later (>10 days). Bioassays on these larvae demonstrated that the speed of kill of CIV carrying AaIT was strikingly enhanced as compared to wild-type CIV. These results suggest that insertion of a toxin gene into CIV provides further opportunities to control a wide range of pest insects, such as weevils, using an iridovirus. (C) 2016 Elsevier Inc. All rights reserved.

Malacosoma neustria (Linnaeus, 1758) (Lepidoptera: Lasiocampidae) causes economic losses in apple, pear, plum, willow, oak and other economically important trees. In this study, an alphabaculovirus was isolated from the larval population of M. neustria from Samsun in the central Black Sea Region of Turkey between 2015 and 2016. Electron microscope analysis of occlusion bodies (OBs) obtained from dead larvae showed that the nucleocapsids of a new isolate (ManeNPV-T4) are multiply enveloped. The Kimura two-parameter analysis and the phylogenetic tree were performed based on concatenated nucleotide and amino acid sequences of the partial lef-8, lef-9 and polh genes from ManeNPV-T4 isolate compared to those of other 51 baculoviruses. Insecticidal activity tests against third instar M. neustria larvae produced 48 to 100% mortalities. The LC 50 of ManeNPV-T4 was 0.78 x 10 3 OBs/ml in M. neustria . Additionally, the isolate caused mortalities lower than 50% in Spodoptera exigua (Hubner, 1808) (Lepidoptera: Noctuidae) , Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae) , Hyphantria cunea (Drury, 1773) (Lepidoptera: Arctiidae) and Helicoverpa armigera (Hubner, 1805) (Lepidoptera: Noctuidae) larvae. Consequently, the new nucleopolyhedrovirus is infectious on M. neustria larvae and other lepidopterans.

The genome of Chilo iridescent virus (CIV) has two open reading frames (ORFs) with matrix metalloprotease (MMP) domains. The protein encoded by ORF 136R contains 178 amino acids with over 40% amino acid sequence identity to hypothetical metalloproteases of other viruses, and the protein 165R contains 264 amino acids with over 40% amino acid sequence identity to metalloproteases of a large group of organisms, primarily including a variety of Drosophila species. These proteins possess conserved zinc-binding motifs in their catalytic domains. In this study, we focused on the functional analysis of these ORFs. They were cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf9 cells with an N-terminal His tag, and purified to homogeneity at 72 hours postinfection using Ni-NTA affinity chromatography. Western blot analyses of purified 136R and 165R proteins with histidine tags resulted in 24- and 34-kDa protein bands, respectively. Biochemical assays with the purified proteins, performed using azocoll and azocasein as substrates, showed that both proteins have protease activity. The enzymatic activities were inhibited by the metalloprotease inhibitor EDTA. Effects of these proteins were also investigated on Galleria mellonella larvae. Insecticidal activity was tested by injecting the larvae with the virus derived from the AcMNPV bacmid carrying 136R or 165R ORFs. The results showed that the baculoviruses harbouring the iridoviral metalloproteases caused early death of the larvae compared to control group. These data suggest that the CIV 136R and 165R ORFs encode functional metalloproteases. This study expands our knowledge about iridoviruses, describes the characterization of CIV matrix metalloproteinases, and might ultimately contribute to the use of this virus as a research tool.