Comparative analysis of the complete genome sequences of 13 baculoviruses revealed a core set of 30 genes, 20 of which have known functions. Phylogenetic analyses of these 30 genes yielded a tree with 4 major groups: the genus Granulovirus (GVs), the group I and II lepidopteran nucleopolyhedroviruses (NPVs), and the dipteran NPV, CuniNPV. These major divisions within the family Baculoviridae were also supported by phylogenies based on gene content and gene order. Gene content mapping has revealed the patterns of gene acquisitions and losses that have taken place during baculovirus evolution, and it has highlighted the fluid nature of baculovirus genomes. The identification of shared protein phylogenetic profiles provided evidence for two putative DNA repair systems and for viral proteins specific for infection of lymantrid hosts. Examination of gene order conservation revealed a core gene cluster of four genes, helicase, lef-5, ac96, and 38K(ac98), whose relative positions are conserved in all baculovirus genomes.

Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) and Granulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.

The predicted amino acid sequence of a newly identified gene of the insect baculovirus Autographa californica nuclear polyhedrosis virus was similar to several uridine 5'-diphosphate (UDP)-glucuronosyl transferases and at least one UDP-glucosyl transferase. Genetic and biochemical studies confirmed that this gene encodes an ecdysteroid UDP-glucosyl transferase (egt). This enzyme catalyzes the transfer of glucose from UDP-glucose to ecdysteroids, which are insect molting hormones. Expression of the egt gene allowed the virus to interfere with normal insect development so that molting was blocked in infected larvae of fall armyworm (Spodoptera frugiperda).

The nucleotide sequence of the DNA genome of Cydia pomonella granulovirus (CpGV) was determined and analysed. The genome is composed of 123 500 bp and has a GMC content of 45n2 %. It contains 143 ORFs of 150 nucleotides or more that show minimal overlap. Onehundred-and-eighteen (82n5 %) of these putative genes are homologous to genes previously identified in other baculoviruses. Among them, 73 are homologous to genes of Autographa californica nucleopolyhedrovirus (AcMNPV), whereas 108 and 98 are homologous to genes of Xestia c-nigrum GV (XcGV) and Plutella xylostella GV (PxGV), respectively. These homologues show on average 37n4 % overall amino acid sequence identity to those from AcMNPV and 45 % to those from XcGV and PxGV. The CpGV gene content was compared to that of other baculoviruses. Several genes reported to have major roles in baculovirus biology were not found in the CpGV genome, such as gp64, the major budded virus glycoprotein gene in some nucleopolyhedroviruses, and lef-7, involved in DNA replication. However, the CpGV genome encodes the large and small subunits of ribonucleotide reductase, three inhibitor of apoptosis (iap) homologues and two protein tyrosine phosphatases. The CpGV, PxGV and XcGV genomes present a noticeably high level of conservation of gene order and orientation. A striking feature of the CpGV genome is the absence of typical homologous repeat sequences. However, it contains one major repeat region and 13 copies of a single 73-77 bp imperfect palindrome.

If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree showed a clear division of the baculoviruses according to the order of their hosts. This division highlighted the need to reconsider the classification of the baculoviruses to include one or possibly two new genera. Furthermore, the specialization of distinct virus lineages to particular insect orders suggests ancient coevolutionary interactions between baculoviruses and their hosts.Coevolution, reciprocal evolution in interacting species driven by natural selection (54), is a major driving factor in the historical associations between pathogens and their hosts (13,25,59). Studies on the evolution of pathogen virulence and host resistance have shown that within populations both pathogens and hosts are able to adapt in response to the interactions (51, 59). However, there is much debate on how these microevolutionary scale changes can influence the patterns of speciation of the interacting species at macroevolutionary levels. Coevolution need not necessarily lead to the cospeciation of the interacting species. However, coevolutionary theories (54) all support the hypothesis that the processes of coadaptations would lead to a general trend of parasite specialization for their hosts (53), regardless of the age of the association. Retroviruses and herpesviruses, with their vertebrate hosts, are good examples of specialist pathogens for which coevolution leading to a certain level of cocladogenesis within subfamilies has been demonstrated (38-40).The family Baculoviridae comprises a diverse group of arthropod-specific DNA viruses. They have been reported worldwide from over 600 host species (37), mostly from insects of the order Lepidoptera but also from the orders Diptera, Hymenoptera, and the crustacean order Decapoda (6, 16). The family Baculoviridae is currently subdivided into two genera based on several criteria, including the morphology of the occlusion bodies (OBs) and on mechanisms of nucleocapsid envelop-

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Autographa californica nucleopolyhedrovirus (AcMNPV), the type member of the virus family Baculoviridae, infects pest insects and has been the subject of many studies for its development as a biopesticide. It is also the virus upon which most of the commercial baculovirus protein expression systems are based. AcMNPV infection of cultured host Spodoptera frugiperda (Sf9) cells can induce a number of alterations of host cell properties including altering the cellular cytoskeleton, an arrest of the cell cycle in G 2 /M, and the global shutoff of host protein translation. Additionally, several cellular transcripts have been shown to be down-regulated following AcMNPV infection. In this study, we take a differential display approach to address whether a global down-regulation of Sf9 host transcripts occurs at late times of infection. Additionally, we also use this approach to search for host mRNAs which are up-regulated at early times of infection, and may be important for facilitating baculovirus infection. From these experiments we can confirm a global down-regulation of Sf9 mRNA levels at late times of infection. We also found that up-regulation of individual host gene RNA levels at early times of infection did not occur frequently. One host transcript which was found to be transiently up-regulated as a result of AcMNPV infection was an Sf9 Hsc70 gene. Hsc70 proteins have been shown to play a vital role in the life-cycle of other large DNA viruses, which suggests that this protein is also important for baculovirus infection.