The spheroidin of an Entomopoxvirus isolated from the grasshopper Anacridium aegyptium (AaEPV) shares low homology with spheroidins from lepidopteranor coleopteran EPVs

Based on virion morphology, the current virus taxonomy groups entomopoxviru ses (EPVs) (Poxvirus: Entomopoxvirinae) from coleopteran and dipteran hosts in separated genera, wilts it keeps viruses infecting either lepidopteran or orthopteran hosts in the same genus. In contrast to the morphological cr iteria, the few data available from recent studies at the genetic level hav e suggested that EPVs infecting different insect orders are phylogeneticall y distant. In order to elucidate EPVs phylogeny we have cloned and sequence the highly conserved/highly expressed spheroidin gene of Anacridium aegypt ium entomopoxvirus (AaEPV). This gene and its promoter is of interest for t he development of genetic engineering on EPVs. The spheroidin gene was loca ted in the AaEPV genome by Southern blot and hybridisation with specific de generated oligonucleotides probes synthesised after partial sequencing of t he purified spheroidin protein. A total of 3489 bp were sequenced. This seq uence included the coding and promoter region of 969 residues 108.8 kDa pro tein identified as spheroidin. AaEPV spheroidin contains 21 cysteine residu es (2.2%) and 14 N-glycosylation putative sites distributed along the seque nce. The cysteine residues are particularly abundant at the C-terminal end of the protein, with 11 residues in the last 118 aa. Our results confirm th at the spheroidin is highly conserved only between EPVs isolated from the s ame insect order. Polyclonal antibodies raised against AaEPV spherules spec ifically revealed spheroidin in Western Blots failing to cross-react with M mEPV or AmEPV spheroidins or MmEPV fusolin. Comparison of spheroidins at th e aa level demonstrate that AaEPV spheroidin shares only 22.2 and 21.9% ide ntity with the lepidopteran AmEPV and the coleopteran MmEPV spheroidins, re spectively, but 82.8% identity with the orthopteran MsEPV spheroidin. Only two highly conserved domains containing the sequence (V/Y)NADTG(C/L) and LF AR(I/A) have been identified in all known spheroidins. The phylogenetic tre e constructed according to the CLUSTALX analysis program revealed that EPVs are clearly separated in three groups - lepidopteran, coleopteran and orth opteran - according to the insect order of the virus hosts. In base to our results, the split of the genus Entomopoxvirus B dissociating lepidopteran and orthopteran EPVs into two different genera is suggested. (C) 2000 Elsev ier Science B.V. All rights reserved.