PCR amplification of the highly conserved baculovirus genes late expression factor 8 (lef-8), late expression factor 9 (lef-9) and polyhedrin/granulin (polh/gran) combined with molecular phylogenetic analyses provide a powerful tool to identify lepidopteran-specific baculoviruses and to study their diversity. In the present investigation, we have improved the degenerate oligonucleotides and corroborated the approach that was recently described by Lange et al. (Lange, M., Wang, H., Zhihong, H., Jehle, J.A., 2004. Towards a molecular identification and classification system of lepidopteran-specific baculoviruses. Virology 325, 36-47.). Baculovirus DNA was isolated from 71 uncharacterized historic baculovirus samples, and partial gene sequences were amplified by using gene-specific degenerate PCR primers. The obtained PCR products were directly sequenced, and the deduced amino acid sequences were compiled and aligned with published sequences of these target genes. A phylogenetic tree of 117 baculoviruses was inferred using maximum parsimony and distance methods. Based on the comprehensive phylogenetic analysis of the partial lef-8, lef-9 and polh/gran genes, we propose a phylogenetic species criterion for lepidopteran-specific baculoviruses that uses the genetic distances of these genes for species demarcation.

Virus genomics provides novel approaches for virus identification and classification. Based on the comparative analyses of sequenced lepidopteran-specific baculovirus genomes, degenerate oligonucleotides were developed that allow the specific amplification of several regions of the genome using polymerase chain reaction (PCR) followed by DNA sequencing. The DNA sequences within the coding regions of three highly conserved genes, namely polyhedrin/granulin (polh/gran), late expression factor 8 (lef-8), and late expression factor 9 (lef-9), were targeted for amplification. The oligonucleotides were tested on viral DNAs isolated from historical field samples, and amplification products were generated from 12 isolated nucleopolyhedrovirus (NPV) and 8 granulovirus (GV) DNAs. The PCR products were cloned or directly sequenced, and phylogenetic trees were inferred from individual and combined data sets of these three genes and compared to a phylogeny, which includes 22 baculoviruses using a combined data set of 30 core genes. This method allows a fast and reliable detection and identification of lepidopteran-specific NPVs and GVs. Furthermore, a strong correlation of the base composition of these three genome areas with that of the complete virus genome was observed and used to predict the base composition of uncharacterized baculovirus genomes. These analyses suggested that GVs have a significantly higher AT content than NPVs.

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