Transcriptional analysis of the putative glycosyltransferase gene (amv248) of the Amsacta moorei entomopoxvirus.

Amsacta moorei entomopoxvirus (AMEV), the most studied member of the genus Betaentomopoxvirus, was initially isolated from Red Hairy caterpillar larvae, Amsacta moorei. According to genome sequence and previous studies it was shown that amv248 encodes a putative glycosyltransferase that is the only conserved attachment protein in betaentomopoxviruses. Transcriptional analysis of the amv248 gene by RT-PCR and qPCR showed that transcription starts at 6h post infection (hpi). Also, transcription was not affected by a DNA replication inhibitor but was severely curtailed by a protein synthesis inhibitor. These results indicate that amv248 belongs to the intermediate class of gene expression. 5' and 3' untranslated regions analysis revealed that transcription initiates at position -126 relative to the translational start site, and ends between 50 and 83 bases after the stop codon. To narrow down the size and location of the gene's promoter, the upstream region as well as several different sized deletions thereof were generated and cloned upstream of a luciferase reporter gene. The constructs were used to measure the Firefly and Renilla luciferase activities in dual assays. The results showed that luciferase activity decreased when bases -198 to -235 of amv248 upstream region were missing. Sequence analysis among the intermediate gene promoters of AMEV showed that TTTAT(T/A)TT(T/A)2TTA is possibly a common motif, however, further investigations are needed to confirm this conclusion.

Comparison of pathogens infection level in Ips typographus (Coleoptera: Curculionidae) beetles sampled in pheromone traps and at place of overwintering.

The importance of pathogens in the population dynamics of Ips typographus remains a subject of ongoing debate. The main objective of our experiment was to compare the pathogen infection levels of individuals overwintering in bark with the levels of individuals from the same population captured with pheromone traps and thereby to determine primary answers as to whether it can be confirmed that pathogenic organisms affect the flight ability of bark beetles or their ability to leave their places of overwintering. A total of 402 I. typographus individuals were analyzed at a study location under limited management. Three pathogens were confirmed to be present: the gregarine Gregarina typographi, the virus ItEPV, and the microsporidium Nosema typographi. Infection levels of Gregarina typographi and ItEPV were the same in beetles collected at places of overwintering and in those beetles collected in pheromone traps within the immediate vicinity. As these pathogens infect the host's intestine, the tendency to leave the places of overwintering is apparently not diminished. A similar analysis and comparison of pathogens located in the fat body might bring different results, as our study only detected N. typographi in a single dissected adult spruce bark beetle.

Purification and partial characterization of an entomopoxvirus (DLEPV) from a parasitic wasp of tephritid fruit flies.

An insect poxvirus [entomopoxvirus (EPV)] occurs in the poison gland apparatus of female Diachasmimorpha longicaudata, a parasitic wasp of the Caribbean fruit fly, Anastrepha suspensa and other tephritid fruit flies. The DlEPV virion is 250-300 nm in diameter, has a "bumpy" appearance and a unipartite double stranded DNA genome of 290-300 kb. DlEPV DNA restriction fragment profiles differed from those reported for Amsacta moorei EPV (AmEPV) and Melanoplus sanguinipes EPV (MsEPV), the only two EPVs whose genomes have been sequenced, and from those reported for vaccinia (Vac), a vertebrate poxvirus (chordopoxvirus, ChPV). Blast search and ClustalW alignment of the amino acids deduced from the 2316 nucleotides of a DlEPV DNA fragment cloned from an EcoR1 genomic library revealed 75-78% homology with the putative DNA-directed RNA polymerases of AmEPV, MsEPV, and two ChPV homologs of the Vac J6R gene. Of the deduced 772 amino acids in the DlEPV sequence, 28.4% are conserved/substituted among the four poxviruses aligned, 12.9% occur in at least one EPV, 6.5% in at least one ChPV, 3.1% in at least one EPV and one ChPV, and 49.1% occur only in DlEPV. Although the RI-36-1 fragment represents a portion of the gene, it contains nucleotides that encode the NADFDGDE consensus sequence of known DNA-directed RNA polymerases. Western blots using a mouse polyclonal anti-DlEPV serum recognized six major protein bands in combined fractions of sucrose-purified DlEPV, at least one band in homogenates of male and female wasps, and at least two bands in host hemolymph that contained DlEPV virions. A digoxigenin-labeled DlEPV genomic DNA probe recognized DNA in dot-blots of male and female wasps. These results confirm that DlEPV is a true EPV and probably a member of the Group C EPVs. Unlike other EPVs, DlEPV does not express the spheroidin protein. Since it also replicates in both the wasp and fly, members of two different insect Orders, DlEPV may represent a new EPV Group, or a subgroup of the Group C viruses.

Entomopoxvirus of cotton bollworm, Helicoverpa armigera (Hbn.).

Occurrence of an Entomopoxvirus (EPV) from a lepidopteran insect viz;. cotton bollworm, H. armigera (HaEPV) along with gross pathological symptoms is reported for the first time in India. Histopathological study revealed that the fat body being the most favoured site of infection followed by haemocytes and gut epithelium. HaEPV was found to be not cross infective to six of the agricultural lepidopteran insect pests except for the potato black cutworm, Agrotis segetum registering 100% mortality showing typical symptom. Further, safety of HaEPV was shown against beneficial insect like mulberry silkworm, Bombyx mori and an useful insect general predator, Chrysoperla carnea.

A new entomopoxvirus from a cockroach: light and electron microscopy.

The cockroach entomopoxvirus caused a chronic infection in cultures of the German cockroach Blattella germanica. Heavily infected specimens showed a reduced mobility. Ellipsoid virus occlusion bodies (8 x 5 to 19 x 12 microm) were found intracellularly in tracheole cells, in the hypodermis, in fat body cells, and in muscles. Several hundred virus particles were integrated in a single occlusion body (OB), their long axis being oriented axially. Ovoid viroids measured 320 x 190 nm and possessed a unilateral, concave core and one lateral body. Starting occlusion, small granules attached to the virus particles which later transformed to a beaded, wavy envelope. An initial halo around the occluded virions disappeared in more central regions of the OB. Virus particles were formed either in a dense cytoplasmic area containing electron-dense viroids, or in a loosely aggregated viroplasm. In the latter, developmental stages were mainly represented by spheres with double membranes enclosing granular material. Spindles and larger crystal-like virus-free inclusion bodies occurred in the cytoplasm. The cytoplasm of infected cells appeared degenerated and the chromatin of the nuclei condensed at the periphery or disintegrated. Taxonomically, the described virus exhibits features of both EPV genus A and EPV genus B. Provisory it is named Blattella germanica EPV (BgEPV). A possible use of the cockroach EPV as a biological control agent is discussed.