Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iri... more Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iridoviridae family. The entirely sequenced dsDNA genome, composed of 212.482 bp, encodes 215 putative open reading frames (ORFs). ORF458R encodes a putative myristoylated membrane protein. RT-PCR analysis of ORF458R expression in the presence of DNA replication and protein synthesis inhibitors showed that this gene is transcribed in the late phase of the virus infection. Time course analysis showed that transcription of ORF458R initiates between 12 and 24 h p.i. and starts to decrease after this point. Transcription of ORF458R initiated 53 nucleotides upstream of the translation start site and ended 40 nucleotides after the stop codon. Dual luciferase reporter gene assay showed that sequences between -61st and +18th nucleotides are essential for promoter activity. Interestingly, a remarkable decrease in promoter activity, in the presence of sequences between -299th and -143rd nucleotides, suggested a repressor activity between these regions. Our results showed that ORF458R is transcriptionally active, and separately located sequences at its upstream region with promoter and repressor activities regulating its expression. This information on the transcriptional analysis of ORF458R will contribute to our understanding of the molecular mechanisms of IIV6 replication.
Background: The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of tr... more Background: The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of trees and forests. Lymantria dispar nucleopolyhedrovirus (LdMNPV) belongs to the Baculoviridae family and is an insect virus specific to gypsy moth larvae. In this study, we describe the complete genome sequences of three geographically diverse isolates, H2 (China), J2 (Japan), and T3 (Turkey), of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Methods: The genomes of isolates H2, J2, and T3 were subjected to shotgun pyrosequencing using Roche 454 FLX and assembled using Roche GS De Novo Assembler. Comparative analysis of all isolates was performed using bioinformatics methods. Results: The genomes of LdMNPV-H2, J2, and T3 were 164,746, 162,249, and 162,614 bp in size, had GC content of 57.25%, 57.30%, and 57.46%, and contained 162, 165, and 164 putative open reading frames (ORFs ≥ 150 nt), respectively. Comparison between the reference genome LdMNPV-5/6 (AF081810) and the genomes...
This chapter focuses on key virus families that affect wild and cultured insect species and descr... more This chapter focuses on key virus families that affect wild and cultured insect species and describes current knowledge on the molecular and ecological aspects of these viruses. Insects can be infected by a variety of viruses each with distinct properties and pathologies. Depending on the taxonomic order to which the insects belong, infections with viruses of certain families are more prominent and strong co-evolutionary relationships between insects and particular virus types are often observed. Viral infections may cause diseases that are often lethal, but viruses can also remain in a persistent or latent stage and only cause a disease outbreak under certain circumstances. We illustrate the impact viruses have on particular groups of insects by providing examples of viral infections in insects in their natural environments, the application of insect viruses in biocontrol programmes, and the effect these viruses have on pollinators. Apart from viruses affecting free-living insects, we also include what is known about virus outbreaks in insects reared on an industrial scale. Furthermore, we include a brief viral disease management section for apiculture and mass rearing of other insects. We conclude this chapter with suggestions on how to obtain the necessary knowledge and tools to better control viral diseases in insects in the future.
Invertebrate iridescent virus 6 (IIV6) is the type species of the Iridovirus genus in the Betairi... more 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.
In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we i... more In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.
ABSTRACT A broad survey of the fall webworm (Lepidoptera: Erebidae) populations in agricultural a... more ABSTRACT A broad survey of the fall webworm (Lepidoptera: Erebidae) populations in agricultural and forested areas in the Central Black Sea Region of Turkey led to the detection of a granulovirus (GV). Hyphantria cunea granulovirus (HycuGV)-Hc1 isolate was characterized and tested against third instar larvae of H. cunea. Electron microscopy confirmed typical GV morphology with ovoid granules of approximately 368 ± 16 nm × 201 ± 19 nm. Each granule contained a single rod-shaped virion with a mean size of 43 ± 12 nm × 250 ± 12 nm. The genome was analyzed by restriction endonuclease and estimated to be ∼112 kb. Partial sequencing of the granulin (gran), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes also confirmed the identity of the virus as HycuGV. A phylogenetic analysis based on these conserved genes, HycuGV-Hc1 grouped together with the previous HycuGV isolate (A5-1) and Estigmene acrea granulovirus (EsacGV) isolate from the same family. The LC50 of HycuGV-Hc1 was 2.6 × 104 occlusion bodies (OBs/ml). Pot experiments, under field conditions, showed significant differences between virus treated and control groups. This is the first study to describe a novel Turkish HycuGV-Hc1 isolate, and preliminary data suggests that the virus has a significant potential as an effective biopesticide for control of H. cunea.
A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa an... more A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 microm in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae.
ABSTRACT Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the ... more ABSTRACT Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol.
Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iri... more Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iridoviridae family. The entirely sequenced dsDNA genome, composed of 212.482 bp, encodes 215 putative open reading frames (ORFs). ORF458R encodes a putative myristoylated membrane protein. RT-PCR analysis of ORF458R expression in the presence of DNA replication and protein synthesis inhibitors showed that this gene is transcribed in the late phase of the virus infection. Time course analysis showed that transcription of ORF458R initiates between 12 and 24 h p.i. and starts to decrease after this point. Transcription of ORF458R initiated 53 nucleotides upstream of the translation start site and ended 40 nucleotides after the stop codon. Dual luciferase reporter gene assay showed that sequences between -61st and +18th nucleotides are essential for promoter activity. Interestingly, a remarkable decrease in promoter activity, in the presence of sequences between -299th and -143rd nucleotides, suggested a repressor activity between these regions. Our results showed that ORF458R is transcriptionally active, and separately located sequences at its upstream region with promoter and repressor activities regulating its expression. This information on the transcriptional analysis of ORF458R will contribute to our understanding of the molecular mechanisms of IIV6 replication.
Background: The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of tr... more Background: The gypsy moth (Lymantria dispar L., Lepidoptera: Erebidae) is a worldwide pest of trees and forests. Lymantria dispar nucleopolyhedrovirus (LdMNPV) belongs to the Baculoviridae family and is an insect virus specific to gypsy moth larvae. In this study, we describe the complete genome sequences of three geographically diverse isolates, H2 (China), J2 (Japan), and T3 (Turkey), of Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Methods: The genomes of isolates H2, J2, and T3 were subjected to shotgun pyrosequencing using Roche 454 FLX and assembled using Roche GS De Novo Assembler. Comparative analysis of all isolates was performed using bioinformatics methods. Results: The genomes of LdMNPV-H2, J2, and T3 were 164,746, 162,249, and 162,614 bp in size, had GC content of 57.25%, 57.30%, and 57.46%, and contained 162, 165, and 164 putative open reading frames (ORFs ≥ 150 nt), respectively. Comparison between the reference genome LdMNPV-5/6 (AF081810) and the genomes...
This chapter focuses on key virus families that affect wild and cultured insect species and descr... more This chapter focuses on key virus families that affect wild and cultured insect species and describes current knowledge on the molecular and ecological aspects of these viruses. Insects can be infected by a variety of viruses each with distinct properties and pathologies. Depending on the taxonomic order to which the insects belong, infections with viruses of certain families are more prominent and strong co-evolutionary relationships between insects and particular virus types are often observed. Viral infections may cause diseases that are often lethal, but viruses can also remain in a persistent or latent stage and only cause a disease outbreak under certain circumstances. We illustrate the impact viruses have on particular groups of insects by providing examples of viral infections in insects in their natural environments, the application of insect viruses in biocontrol programmes, and the effect these viruses have on pollinators. Apart from viruses affecting free-living insects, we also include what is known about virus outbreaks in insects reared on an industrial scale. Furthermore, we include a brief viral disease management section for apiculture and mass rearing of other insects. We conclude this chapter with suggestions on how to obtain the necessary knowledge and tools to better control viral diseases in insects in the future.
Invertebrate iridescent virus 6 (IIV6) is the type species of the Iridovirus genus in the Betairi... more 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.
In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we i... more In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.
ABSTRACT A broad survey of the fall webworm (Lepidoptera: Erebidae) populations in agricultural a... more ABSTRACT A broad survey of the fall webworm (Lepidoptera: Erebidae) populations in agricultural and forested areas in the Central Black Sea Region of Turkey led to the detection of a granulovirus (GV). Hyphantria cunea granulovirus (HycuGV)-Hc1 isolate was characterized and tested against third instar larvae of H. cunea. Electron microscopy confirmed typical GV morphology with ovoid granules of approximately 368 ± 16 nm × 201 ± 19 nm. Each granule contained a single rod-shaped virion with a mean size of 43 ± 12 nm × 250 ± 12 nm. The genome was analyzed by restriction endonuclease and estimated to be ∼112 kb. Partial sequencing of the granulin (gran), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes also confirmed the identity of the virus as HycuGV. A phylogenetic analysis based on these conserved genes, HycuGV-Hc1 grouped together with the previous HycuGV isolate (A5-1) and Estigmene acrea granulovirus (EsacGV) isolate from the same family. The LC50 of HycuGV-Hc1 was 2.6 × 104 occlusion bodies (OBs/ml). Pot experiments, under field conditions, showed significant differences between virus treated and control groups. This is the first study to describe a novel Turkish HycuGV-Hc1 isolate, and preliminary data suggests that the virus has a significant potential as an effective biopesticide for control of H. cunea.
A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa an... more A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 microm in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae.
ABSTRACT Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the ... more ABSTRACT Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol.
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Papers by Remziye Nalcacioglu