Identification of the potential matrix protein of invertebrate iridescent virus 6 (IIV6).

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic virus with a ∼212 kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the IIV6 virion consists of 54 virally encoded proteins. Interactions among the structural proteins were investigated using the yeast two-hybrid system, revealing that the protein of 415R ORF interacts reciprocally with the potential envelope protein 118L and the major capsid protein 274L. This result suggests that 415R might be a matrix protein that plays a role as a bridge between the capsid and the envelope proteins. To elucidate the function of 415R protein, we determined the localization of 415R in IIV6 structure and analyzed the properties of 415R-silenced IIV6. Specific antibodies produced against 415R protein were used to determine the location of the 415R protein in the virion structure. Both western blot hybridization and immunogold electron microscopy analyses showed that the 415R protein was found in virions treated with Triton X-100, which degrades the viral envelope. The 415R gene was silenced by the RNA interference (RNAi) technique. We used gene-specific dsRNA's to target 415R and showed that this treatment resulted in a significant drop in virus titer. Silencing 415R with dsRNA also reduced the transcription levels of other viral genes. These results provide important data on the role and location of IIV6 415R protein in the virion structure. Additionally, these results may also shed light on the identification of the homologs of 415R among the vertebrate iridoviruses.

Janthinobacterium kumbetense sp. nov., a violacein-producing bacterium isolated from spring water in Turkey, and investigation of antimicrobial activity of violacein.

Strain GKT was isolated from the Kumbet plateu of Giresun in Turkey. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GKT belonged to genus Janthinobacterium and 16S rRNA gene sequence similarities with all type strains of the genus Janthinobacterium were 98.89%-99.78%. The calculated pairwise average nucleotide identity (ANI) values between strain GKT and all type strains of Janthinobacterium species were in the range of 79.8%-93.2%. In addition, digital DNA-DNA hybridization (dDDH) values were in the range of 23.0%-51.7%. Major fatty acids are C10:03OH, C12:0, C16:1ω7c, C16:0, and C18:1ω7c, and polar lipids included phosphatidylethanolamine, phosphatidylglycerol, also one unidentified phospholipid and one unidentified aminophospholipid. The respiratory quinone of strain GKT was determinated to be Q-8. The genome sizes of strain GKT was 6 197 538 bp with 63.16% G + C ratio. Strain GKT is Gram-stain-negative, aerobic, rod-shaped, and motile. A violet pigment was produced by strain GKT. The crude violacein pigments were separated into three diferent bands on a TLC sheet. Then violacein and deoxyviolacein were purifed by vacuum liquid column chromatography and identifed by NMR spectroscopy. The antimicrobial activities of purifed violacein and deoxyviolacein were screened for seven microorganisms. Based on the results of the morphological, biochemical, physiological, phylogenetic, and genomic characteristics, we propose classifying the strain GKT as representative of a novel species of the genus Janthinobacterium, for which the name Janthinobacterium kumbetense sp. nov. is proposed (GKT = LMG 32662T = DSM 11423T).

Genome Sequencing and Organization of Three Geographically Different Isolates of Nucleopolyhedrovirus from the Gypsy Moth Reveal Significant Genomic Differences.

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 of LdMNPV-H2, J2, and T3 revealed differences in gene content. Compared with LdMNPV-5/6, ORF5, 6, 8, 10, 31, and 67 were absent in LdMNPV-H2, ORF5, 13, and 66 were absent in LdMNPV-J2, and ORF10, 13, 31, and 67 were absent in LdMNPV-T3. In addition, the gene encoding the mucin-like protein (ORF4) was split into two parts in isolates H2 and T3 and designated ORF4a and ORF4b. Phylogenetic analysis grouped isolates H2 and J2 in a different cluster than isolate T3, which is more closely related to the Turkish and Polish isolates. In addition, H2 was found to be closely related to a South Korean LdMNPV isolate. Conclusion: This study provided a more detailed overview of the relationships between different geographic LdMNPV isolates. The results showed remarkable differences between groups at the genome level.

Conserved motifs in the invertebrate iridescent virus 6 (IIV6) genome regulate virus transcription.

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.

Genome sequence analysis of a Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR) isolated from Heliothis peltigera in Turkey.

The entire genome of Helicoverpa armigera single nucleopolyhedrovirus (HearNPV-TR) was sequenced, and compared to genomes of other existing isolates. HearNPV-TR genome is 130.691 base pairs with a 38.9% G+C content and has 137 open reading frames (ORFs) of ≥ 150 nucleotides. Five homologous repeated sequences (hrs) and two baculovirus repeated ORFs (bro-a and bro-b) were identified. Phylogenetic analysis showed that HearNPV-TR is closer to HaSNPV-C1, HaSNPV-G4, HaSNPV-AU and HasNPV. However, there are significant differences in hr3, hr5 regions and in bro-a gene. Pairwise Kimura-2 parameter analysis of 38 core genes sequences of HearNPV-TR and other Helicoverpa NPVs showed that the genetic distances for these sequences were below 0.015 substitutions/site. Genomic differences as revealed by restriction profiles indicated that hr3, hr5 regions and bro-a gene may play a role in the virulence of HearNPV-TR.

Genome sequence analysis and organization of the Hyphantria cunea granulovirus (HycuGV-Hc1) from Turkey.

The fall webworm (Hyphantria cunea) impacts a wide variety of crops and cultivated broadleaf plant species. The pest is native to North America, was introduced to Europe and has since spread further as far as central Asia. Despite several attempts to control its distribution, the pest continues to spread causing damage all over the world. A naturally occurring baculovirus, Hyphantria cunea granulovirus (HycuGV-Hc1), isolated from the larvae of H. cunea in Turkey appears to have a potential as microbial control agent against this pest. In this report we describe the complete genome sequence and organization of the granulovirus isolate (HycuGV-Hc1) that infects the larval stages and compare it to other baculovirus genomes. The HycuGV-Hc1 genome is a circular double-stranded DNA of 114,825 bp in size with a nucleotide distribution of 39.3% G + C. Bioinformatics analysis predicted 132 putative open reading frames of (ORFs) ≥ 150 nucleotides. There are 24 ORFs with unknown function. Seven homologous repeated regions (hrs) and two bro genes (bro-1 and bro-2) were identified in the genome. Comparison to other baculovirus genomes, HycuGV-Hc1 revealed some differences in gene content and organization. Gene parity plots and phylogenetics confirmed that HycuGV-Hc1 is a Betabaculovirus and is closely related to Plutella xylostella granulovirus. This study expands our knowledge on the genetic variation of HycuGV isolates and provides further novel knowledge on the nature of granuloviruses.

A new Helicoverpa armigera Nucleopolyhedrovirus isolate from Heliothis peltigera (Denis & Schiffermuller) (Lepidoptera: Noctuidae) in Turkey.

This study reports a new Helicoverpa armigera nucleopolyhedrovirus (NPV) isolated from Heliothis peltigera (Denis & Schiffermuller), collected in the vicinity of Adana, Turkey. Infection was confirmed by tissue polymerase chain reaction and sequence analysis. Results showed that dead H. peltigera larvae contain Helicoverpa armigera nucleopolyhedrovirus. Thus, the isolate was named as HearNPV-TR. Microscopy studies indicated that occlusion bodies were 0.73 to 1.66 µm in diameter. The nucleocapsids are approximately 184 × 41 nm in size. The genome of HearNPV-TR was digested with KpnI and XhoI enzymes and calculated as 130.5 kb. Phylogenetic analysis showed that HearNPV-TR has close relation with the H. armigera SNPV-1073 China isolate. The Kimura analysis confirmed that the isolate is a variant of H. armigera NPV. Bioassays were performed using six different concentrations (1 × 103 to 1 × 108 occlusion bodies (OBs)/mL) on 2nd instar larvae of H. peltigera, H. armigera, Heliothis viriplaca, Heliothis nubigera. LC50 values were calculated to be 9.5 × 103, 1.9 × 104, 8.6 × 104 and 9.2 × 104 OBs/mL within 14 days, respectively. Results showed that it is a promising biocontrol agent against Heliothinae species.

Chilo iridescent virus encodes two functional metalloproteases.

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.

Complete genome sequence analysis of the Malacosoma neustria nucleopolyhedrovirus from Turkey.

The lackey moth, Malacosoma neustria (Linnaeus, 1758), a worldwide pest, causes extensive economic losses particularly on hazelnut, plum, oak, poplar, and willow trees. A baculovirus, Malacosoma neustria nucleopolyhedrovirus (ManeNPV-T2), has been isolated from the larvae collected in Turkey and appears to have a potential as a microbial control agent. In this study, we describe the complete genome sequence of ManeNPV-T2 and compare it to other sequenced baculovirus genomes. The ManeNPV-T2 genome is a circular double-stranded DNA molecule of 130,202 bp, has 38.2% G + C, and is predicted to contain 131 putative open reading frames (ORFs) each with a coding capacity of more then 50 amino acids. There are 27 ORFs with unknown function of which 6 are unique to ManeNPV-T2. Eleven homologous regions (hrs) and two bro genes (bro-a and bro-b) were identified in the genome. There are two homologues of chaB and nicotinamide riboside kinase-1 genes, separated from themselves with a few nucleotides. Additionally, ac145, thought to be per os infectivity factor (pif) gene, is also found as two homologues. All 38 core genes are found in the ManeNPV-T2 genome. The phylogenetic tree of ManeNPV-T2 in relation to 50 other baculoviruses whose genomes have been completely sequenced showed ManeNPV-T2 to be closely related to the group II NPVs. This study expands our knowledge on baculoviruses, describes the characterization ManeNPV, and ultimately contributes to the registration of this virus as a microbial pesticide.

Protein-protein interactions among the structural proteins of Chilo iridescent virus.

Chilo iridescent virus (CIV), officially named invertebrate iridescent virus 6 (IIV6), is a nucleocytoplasmic virus with a ~212-kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the CIV virion consists of 54 virally encoded proteins. In this study, we identified the interactions between the structural proteins using the yeast two-hybrid system. We cloned 47 structural genes into both bait and prey vectors, and then analysed the interactions in Saccharomyces cerevisiae strain AH109. A total of 159 protein-protein interactions were detected between the CIV structural proteins. Only ORF 179R showed a self-association. Four structural proteins that have homologues in iridoviruses (118L, 142R, 274L and 295L) showed indirect interactions with each other. Seven proteins (138R, 142R, 361L, 378R, 395R, 415R and 453R) interacted with the major capsid protein 274L. The putative membrane protein 118L, a homologue of the frog virus 3/Ranagrylio virus 53R protein, showed direct interactions with nine other proteins (117L, 229L, 307L, 355R, 366R, 374R, 378R, 415R and 422L). The interaction between 118L and 415R was confirmed by a GST pull-down assay. These data indicate that 415R is a potential matrix protein connecting the envelope protein 118L with the major capsid protein 274L.

The protein-protein interactions between Amsacta moorei entomopoxvirus (AMEV) protein kinases (PKs) and all viral proteins.

Entomopoxviruses are an important group of viruses infecting only insects. They belong to Poxviridae which infect both invertebrates and vertebrates, including humans. Protein kinases are known to have roles at virus morphogenesis, host selectivity, the regulation of cell division and apoptosis in some vertebrate poxviruses. In this study, 2 protein kinases (PKs) (AMV153 and AMV197) of Amsacta moorei entomopoxvirus (AMEV) were investigated for the interactions among 230 viral proteins using yeast two-hybrid system (Y2H). For this purpose, two protein kinases and 230 viral genes were cloned into the bait and prey vectors, respectively. Bait vectors were introduced into Saccharomyces cerevisiae AH109. Expression of the bait genes were confirmed by western blot analysis. Both yeast strains of bait were transformed individually with each prey clone and grown on a selective medium (minimal synthetic defined) to determine the protein-protein interactions between bait and prey proteins. Transformations identified totally 16 interactions among AMEV protein kinases and all viral proteins of which 5 belong to AMV153 and 11 belong to AMV197. One of the five interactions detected for AMV153 protein kinase is self-association. Its other four interactions are with two virus entry complex proteins (AMV035 and AMV083), a membrane protein (AMV165) and a subunit of RNA polymerase (AMV230). The other protein kinase, AMV197, interacted with two virus entry complex proteins (AMV035 and AMV083) as AMV153, a caspase-2 enzyme (AMV063), a Holliday junction resolvase (AMV162), a membrane protein (AMV165), a subunit of RNA polymerase (AMV230) and five other hypothetical proteins (AMV026, AMV040, AMV062, AMV069, AMV120) encoded by AMEV genome. Glutathione S-transferase (GST) pull-down assay was used to confirm all interactions described by Y2H analysis. In addition, the theoretical structures of the two of 16 interactions were interpreted by docking analysis. Consistent with Y2H and pull down assays, docking analysis also showed the interactions of AMV063 with AMV153 and AMV197. Detected interactions of the AMEV viral proteins with viral protein kinases could lead to the understanding of the regulation of the viral activities of interacted viral proteins.

Chilo iridescent virus (CIV) ORF 012L encodes a protein with both exonuclease and endonuclease functions.

Chilo iridescent virus (CIV) is the type member of the genus Iridovirus within the family Iridoviridae. The virions of CIV contain a single linear dsDNA molecule that is circularly permuted and terminally redundant. The genome of CIV contains an open reading frame (ORF 012L) encoding a protein homologous to exonuclease II of Schizosaccharomyces pombe. In this study, we focused on the characterization of CIV ORF 012L. The target ORF was cloned into the pET28a vector, expressed in E. coli strain BL21 (DE3) pLysS with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified CIV 012L confirmed that this viral protein is a functional 5'-3' exonuclease that digests 3'-biotin-labelled oligonucleotides and linear double-stranded DNA (dsDNA) molecules from their 5' termini in a highly processive manner. CIV 012L also has a potent endonuclease activity on dsDNA in vitro. In addition, CIV 012L converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) and then open circular form into linear form (RFIII). Endonuclease activity of CIV 012L was optimal in the presence of 10 mM Mg(2+) or 30 mM Mn(2+) ions and at 150 mM NaCl or KCl salt concentrations. The highest endonuclease activity was obtained at pH 8, and it reached a maximum at 55 °C. The CIV 012L protein showed deficiencies for both double- and single-stranded RNAs.

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.

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.

In search of pathogens: transcriptome-based identification of viral sequences from the pine processionary moth (Thaumetopoea pityocampa).

Thaumetopoea pityocampa (pine processionary moth) is one of the most important pine pests in the forests of Mediterranean countries, Central Europe, the Middle East and North Africa. Apart from causing significant damage to pinewoods, T. pityocampa occurrence is also an issue for public and animal health, as it is responsible for dermatological reactions in humans and animals by contact with its irritating hairs. High throughput sequencing technologies have allowed the fast and cost-effective generation of genetic information of interest to understand different biological aspects of non-model organisms as well as the identification of potential pathogens. Using these technologies, we have obtained and characterized the transcriptome of T. pityocampa larvae collected in 12 different geographical locations in Turkey. cDNA libraries for Illumina sequencing were prepared from four larval tissues, head, gut, fat body and integument. By pooling the sequences from Illumina platform with those previously published using the Roche 454-FLX and Sanger methods we generated the largest reference transcriptome of T. pityocampa. In addition, this study has also allowed identification of possible viral pathogens with potential application in future biocontrol strategies.

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.

Molecular characterization of chitinase genes from a local isolate of Serratia marcescens and their contribution to the insecticidal activity of Bacillus thuringiensis strains.

The chitinase B (chiB) and C (chiC) genes and flanking regions from a local isolate of Serratia marcescens were cloned individually and sequenced. Results showed that these chiB and chiC genes have a 96 % maximum similarity with chiB and chiC from different S. marcescens species (GenBank numbers Z36295.1 and AJ630582.1, respectively). The amplified chiB fragment, including some upstream and downstream regions, is 1,689-bp long with an open reading frame of 1,500 bp. The amplified fragment of chiC is 1,844 bp with an open reading frame of 1,443 bp. These sequences were submitted to the GenBank with accession numbers JX847796 (chiB) and JX847797 (chiC). Putative promoter regions and Shine-Dalgarno sequences were identified in both genes. The genes were cloned into a shuttle vector and the constructs were designated as pHYSB and pHYSC, respectively. Both plasmids were introduced separately into kurstaki and israelensis strains of Bacillus thuringiensis and the insecticidal activities of the engineered B. thuringiensis strains were assayed in larvae of Galleria mellonella and adult of Drosophila melanogaster. Engineered B. thuringiensis strains showed higher insecticidal activity than parental strain and the parental S. marcescens. In addition, pHYSB and pHYSC were stable over 16 daily passages under non-selective conditions in transformed B. t. israelensis 5724 strain.

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 ß-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 host's 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.

Transcriptomic analysis of Chilo iridescent virus immediate early promoter.

Chilo iridescent virus (CIV) is an insect virus belonging to the Iridoviridae. The DNA genome (212,482 base pairs) is entirely sequenced, however very little is known about viral gene regulation, expression and function. The structure and transcriptional regulation of the CIV 012L gene is investigated in this study. Infection of Bombyx mori SPC-BM-36 cells in the presence of Ara-C (inhibits DNA replication) or cycloheximide (inhibits protein synthesis), followed by RT-PCR on isolated total RNA, showed that CIV 012L is transcribed as an immediate-early gene. Detecting the RNA transcript of the CIV 012L early in infection confirmed the data about the temporal class of the gene obtained with the inhibitors. Time course transcription of the gene showed that the transcription starts immediately after infection and reach up to maximum level at 4h p.i. 5' RACE analysis on RNA isolated from CIV-infected BM cells showed that the transcription initiation site is located 30 nucleotides upstream of the translational start codon. To map the limits of the putative promoter of this gene, upstream sequences of various lengths were cloned in front of a firefly luciferase reporter gene. The resulting plasmid constructs were tested in a transfection assay, in which the baculovirus IE-l promoter fused to Renilla luciferase was used as an internal control for transfection efficiency. A gradual reduction in luciferase expression occurred as the deletions extended from -200 to -10, relative to the transcription start site. It is clearly shown that sequences between -20 and -10 relative to the transcription start site have key promoter activity for CIV 012L gene. However this key sequence could not be found at the upstream region of CIV's other potential immediate early genes.

Amsacta moorei entomopoxvirus encodes a functional DNA photolyase (AMV025).

The major damage induced in DNA by ultraviolet light is the induction of cyclobutane pyrimidine dimers (CPDs). Amsacta moorei entomopoxvirus (AMEV) encodes a CPD photolyase (AMV025) with a putative role in converting these dimers back into monomers. In infected Lymantria dispar cells transcription of the AMV025 gene started 8h post inoculation (p.i.) and continued through 38hp.i. Transcription was inhibited by a DNA synthesis blocker. Transient expression in an Escherichia coli strain that lacks its endogenous photolyase, rescued growth of the UV-irradiated bacteria in a light-dependent manner, showing that AMV025 encodes a functional DNA photolyase.