Sequence and organization of the Heliothis virescens ascovirus genome Sassan Asgari, 1 John Davis, 2 David Wood, 2 Peter Wilson 2 and Annette McGrath 2 Correspondence Sassan Asgari s.asgari@uq.edu.au 1 School of Integrative Biology, University of Queensland, St Lucia, QLD 4072, Australia 2 Australian Genome Research Facility, University of Queensland, St Lucia, QLD 4072, Australia Received 18 October 2006 Accepted 30 November 2006 The nucleotide sequence of the Heliothis virescens ascovirus (HvAV-3e) DNA genome was determined and characterized in this study. The circular genome consists of 186 262 bp, has a G+C content of 45.8 mol% and encodes 180 potential open reading frames (ORFs). Five unique homologous regions (hrs), 23 ‘baculovirus repeat ORFs’ (bro) and genes encoding a caspase homologue and several enzymes involved in nucleotide replication and metabolism were found in the genome. Several ascovirus (AV)-, iridovirus- and baculovirus-homologous genes were identified. The genome is significantly larger than the recently sequenced genomes of Trichoplusia ni AV (TnAV-2c) and Spodoptera frugiperda AV (SfAV-1a). Gene-parity plots and overall similarity of ORFs indicate that HvAV-3e is related more closely to SfAV-1a than to TnAV-2c. INTRODUCTION Ascoviruses (AVs) are double-stranded DNA (dsDNA) viruses that cause a chronic infection in lepidopterans, mainly members of the family Noctuidae with economic significance (Federici et al., 2005). AVs have large, enveloped virions (1306400 nm) that are allantoid to bacilliform in shape and have a complex symmetry (Federici et al., 1991). The size of the circular genome ranges from 100 to 180 kbp (Bigot et al., 1997b; Cheng et al., 1999; Federici et al., 2005). The pathology of AVs is distinguished from that of other viruses by producing a milky-white discoloration of the haemolymph in the infected host, caused by accumulation of a high concentration of virion-containing vesicles. The vesicles are formed by the cleavage of the host-cell membrane, a unique sign of AV infection (Federici, 1983). Four species of AV are currently recognized: Spodoptera frugiperda AV (SfAV-1), Trichoplusia ni AV (TnAV-2), Heliothis virescens AV (HvAV-3) and Diadromus pulchellus AV (DpAV-4). Phylogenetic studies based on several genes characterized from the AVs indicate that SfAV-1, TnAV-2 and HvAV-3 cluster together on the same branch, whereas DpAV is found on a separate branch (Federici & Bigot, 2003). This is probably a reflection of the biology and association of the viruses with their parasitoid vector. Members of the former group are transferred mechanically by parasitoids and are antagonistic, leading to the death of the developing parasitoid (Stasiak et al., 2005), whereas DpAV is transmitted vertically and has a mutualistic association with its wasp vector (Bigot et al., 1997a). Further phylogenetic studies revealed that invertebrate iridoviruses are related closely to AVs (Stasiak et al., 2003), despite major differences in their cell biology. An Australian AV was isolated from Helicoverpa armigera larvae in south-east Queensland (Newton, 2003). Here, by using DNA hybridization, we show that little hybridization occurs between TnAV-2 and the Australian isolate under high-stringency conditions. However, HvAV-3 genomic DNA (gDNA) hybridized strongly to all restriction frag- ments from the Australian isolate. This isolate was previous- ly designated HvAV-3e, a variant of HvAV, based only on the DNA polymerase and the major capsid protein sequences (Stasiak et al., 2005). The hybridization results confirmed the identity of the isolate as a variant of HvAV-3. Recently, the complete genome sequences of SfAV-1a (156 922 bp) and TnAV-2c (174 059 bp) were determined (Bideshi et al., 2006; Wang et al., 2006). TnAV is very AT- rich (G+C content, 35.4 mol%) and contains two homo- logous regions (hrs), whereas SfAV has a G+C content of 49.2 mol% with four complete repeats and a partial one. Here, we present the complete genome sequence of HvAV- 3e, with a G+C content of 45.8 mol%. The genome is significantly larger than those of TnAV-2c and SfAV-1a, with five hr regions containing transposase domains that are not present in the TnAV-2c genome. In addition, the HvAV- 3e genome contains 23 baculovirus repeated ORF (Bro) proteins, compared with three in TnAV-2c and seven in SfAV-1a. Only one copy of the major capsid protein and thymidine kinase were found in HvAV-3e, compared with two copies of each gene in TnAV-2c. The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is EF133465. 1120 0008-2651 G 2007 SGM Printed in Great Britain Journal of General Virology (2007), 88, 1120–1132 DOI 10.1099/vir.0.82651-0