Remziye Nalcacioglu

Open reading frame 193R of Chilo iridescent virus encodes a functional inhibitor of apoptosis (IAP)

Programmed cell death or apoptosis is a major defense mechanism in insects in response to viral infections. The genome of Chilo iridescent virus (CIV) has three ORFs with homology to baculovirus inhibitor of apoptosis (iap) genes. The proteins encoded by the 157L, 193R, and 332L ORFs contain 152, 208 and 234 amino acids, respectively. While all three proteins contain C-terminal RING domains, only the protein encoded by ORF 193R contains a baculoviral iap repeat (BIR) domain, indicative of a putative IAP protein. The 193R protein has 28 and 27% similarity in amino acid sequence to the Orgyia pseudotsugata MNPV and Cydia pomonella granulovirus IAP-3 proteins, respectively. ORF 193R from CIV is the only gene known to exist among the Iridoviridae that encodes a BIR domain. 193R is transcribed early during CIV infection, and its transcription is not dependent on the synthesis of early viral proteins. When this putative CIV IAP was transiently expressed in SPC-BM-36 and Sf21 cells under the control of an immediate early baculovirus promoter it significantly reduced apoptosis induced by actinomycin-D. Silencing of the CIV iap gene (193R) in CIV infected SPC-BM-36 cells with 193R-specific dsRNA resulted in apoptosis. Thus, CIV ORF 193R is the first iap gene identified in an iridovirus, which encodes a functional IAP protein.

The relationship between insecticidal effects and chitinase activities of Coleopteran-originated entomopathogens and their chitinolytic profile

Peritrophic membrane (PM) is present in most insects’ midgut and acts as a mechanical barrier to protect the epithelium from various harmful factors such as pathogens or toxins. Chitinase is a virulence factor due to its ability to degrade the chitin content of PM. Therefore, chitinase is a mediator for easier binding of toxins to gut epithelium and intercepting nutritional absorption in the midgut. One hundred and eight bacterial isolates derived from microbial flora of coleopteran pests were screened to determine chitin-producing entomopathogenic bacteria. The M9 chitin–-agar method and polymerase chain reaction with specific primers for a conserved domain of chitinase genes indicated that 23 of the 108 isolates have chitinase activity. The chitinase activities of the chitinase-positive bacteria were measured. We compared these results with the insecticidal activities results to determine, statistically, the potential relationship between the chitinase activities and the insecticidal activity. Consequently, 21.3% of bacterial isolates showed chitinolytic ability and among these the chitinase-positive bacteria, Serratia marcescens, was found the most active one in the M9-CAD method. More importantly, our study indicated a very strong positive correlation between the insecticidal activities of isolates and the chitinase activities with the M9-CAD method (r2 = 0.96, p ≤ 0.01), but not with the DNS method (r2 = −0.279, p ≤ 0.01). This strong relationship of entomopathogens has a high potential for biocontrol of Coleopteran pests.

Highly pathogenic Bacillus thuringiensis subsp. tenebrionis from European shot-hole borer, Xyleborus dispar (Coleoptera: Scolytidae)

Abstract The entomopathogenic bacterium Bacillus thuringiensis is the most widely used biopesticide. In this study, to find and identify the more toxic B. thuringiensis strains against coleopteran pests, we isolated a B. thuringiensis strain (Xd3) from European shot‐hole borer, Xyleborus dispar (Coleoptera: Scolytidae), a higly damaging pest of hazelnut. Based on various morphological, physiological, biochemical, and molecular characteristics, the bacterial isolate was identified as B. thuringiensis subsp. tenebrionis (morrisoni) serovar H8a8b. This isolate was compared with the reference strains by scanning electron microscopy, SDS‐PAGE analysis, cry gene content, and insecticidal activity. Isolate Xd3 forms a flat‐square inclusion containing a protein component of c. 70 kDa. PCR analysis showed that the Xd3 has a cry gene, cry3. Toxicity tests were performed against coleopteran species. One hundred percent mortality was observed against larvae of Agelastica alni (Coleoptera: Chrysomelidae). The others were 90% for Amphimallon solstitiale (Coleoptera: Scarabaeidae), and Melolontha melolontha (Coleoptera: Scarabaeidae). Our results indicate that B. thuringiensis subsp. tenebrionis (Xd3) may be valuable as biological control agent for coleopteran insects.

Construction and Characterization of a Recombinant Invertebrate Iridovirus

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.

Enhanced insecticidal activity of Chilo iridescent virus expressing an insect specific neurotoxin.

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-Δ157L-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-Δ157L/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-Δ157L/gfp-AaIT and rCIV-Δ157L-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-Δ157L/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 (>10days). 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.

Characterization of a nucleopolyhedrovirus variant of the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae) in Turkey

ABSTRACT The gypsy moth (Lymantria dispar L., Lepidoptera: Lymantriidae) is one of the most serious pest of various forestal, food and industrial crops worldwide. We have characterized a new Lymantria dispar nucleopolyhedrovirus (LdMNPV-T4) variant, which was isolated from dead L. dispar larvae in Turkey. Scanning electron microscope observations showed that the polyhedral occlusion bodies (OBs) of the LdMNPV-T4 were irregularly shaped. Transmission electron microscopy revealed that OBs of LdMNPV-T4 were occupied with several virions in which multiple nucleocapsids packaged by viral envelope. Restriction analysis of the LdMNPV-T4 DNA purified from the viral inclusion bodies yielded BamHI, BglII, EcoRI and HindIII fragments. The mean size estimated for the complete LdMNPV-T4 genome was calculated to be 163.3 kb. Phylogenetic analysis of amplified polh, lef-8 and lef-9 sequences showed its relation to the other NPVs from Lymantria species. Mortality values of the LdMNPV-T4 at four different concentrations against third instar larvae of L. dispar ranged from 45% to 88%. These results suggest that LdMNPV-T4 isolated from Turkey is a promising microbial control agent to be utilized for the biological control of L. dispar.

Induction of apoptosis by the Amsacta moorei entomopoxvirus

CF-70-B2 cells derived from the spruce budworm (Choristoneura fumiferana) undergo apoptosis when infected with Amsacta moorei entomopoxvirus (AMEV), as characterized by membrane blebbing, formation of apoptotic bodies, TdT-mediated dUTP nick-end labelling (TUNEL) staining, condensed chromatin and induction of caspase-3/7 activity. The apoptotic response was reduced when cells were infected with UV-inactivated AMEV, but not when infected in the presence of the DNA synthesis inhibitor, cytosine β-d-arabinofuranoside. Hence, only pre-DNA replication events were involved in inducing the antiviral response in CF-70-B2 cells. The virus eventually overcame the hosts antiviral response and replicated to high progeny virus titres accompanied by high levels of caspase-3/7 activity. The CF-70-B2 cells were less productive of progeny virus in comparison to LD-652, a Lymantria dispar cell line routinely used for propagation of AMEV. At late stages of infection, LD-652 cells also showed characteristics of apoptosis such as oligosomal DNA fragmentation, TUNEL staining, condensed chromatin and increased caspase-3/7 activity. Induction of apoptosis in LD-652 cells was dependent on viral DNA replication and/or late gene expression. A significantly reduced rate of infection was observed in the presence of general caspase inhibitors Q-VD-OPH and Z-VAD-FMK, indicating caspases may be involved in productive virus infection.

The Biology of Chilo Iridescent Virus

Chilo iridescent virus (CIV) is the type species for genus Iridovirus, and belongs to the family Iridoviridae. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The genome of CIV has been entirely sequenced. The CIV virion consists of an unusual three-layer structure containing an outer proteinaceous capsid, an intermediate lipid membrane, and a core DNA-protein complex containing the genome. CIV has a broad host spectrum and has, in general, a limited mortality effect on its hosts. Up to now there have been several studies about CIV describing its structure, ecology, and molecular biology. In this review study we present all these studies together to describe the CIV.

Characterisation of three Alphabaculovirus isolates from the gypsy moth, Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

ABSTRACT In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170 kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 103 and 106 OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC50 and LC95 values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC50 value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.

Genome-wide analysis of differential mRNA expression of Amsacta moorei entomopoxvirus, mediated by the gene encoding a viral protein kinase (AMV197).

Insect-born entomopoxviruses (Fam: Poxviridae) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus. Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha (ef1a) gene) was downregulated in the absence of amv197. Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process.