No abstract

The Musca domestica salivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 39-ends (39-RACE) and RT-PCR. Direct sequencing of 39-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 39-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 39-RACE or RT-PCR. A series of 59-RACE reactions were conducted on selected ORFs that were identified by 39-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, the downstream ORFs were detected as single transcripts as well as components of the tandem transcripts, whereas the upstream ORFs were found only in tandem transcripts. The only exception was the upstream ORF MdSGHV084, which was differentially transcribed as a single transcript at 1 and 2 days post-infection (days p.i.) and as a tandem transcript (MdSGHV084/085) at 2 days p.i. Transcriptome analysis of MdSGHV detected splicing in the 39 untranslated region (39-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 39-RACE products without cloning served as an alternative approach to detect both 39-UTRs and transcript variants of this large DNA virus. INTRODUCTIONSalivary gland hypertrophy viruses (SGHVs) have been detected in several dipterans, including the house fly (Musca domestica), tsetse fly (Glossina spp.), and the narcissus bulb fly (Merodon equestris) (Amargier et al., 1979;Coler et al., 1993;Gouteux, 1987;Jaenson, 1978;Minter-Goedbloed & Minter, 1989;Otieno et al., 1980;Shaw & Moloo, 1993). Infection by these viruses both induces SGH and reduces reproductive fitness (Lietze et al., 2007;Sang et al., 1998Sang et al., , 1999. Currently, our lab is using the M. domestica SGHV (MdSGHV) to study the replicative pathway and mode of action of this unique virus group. Significantly, MdSGHV is capable of pervasive development in adult salivary glands; 100 % of the glands display SGH and release copious levels of infectious virus between 48 and 72 h post-injection (V. U. Lietze & D. G. Boucias, unpublished results). The synchronized infection displayed by this virus and the access to virus-free house fly colonies provides an inexpensive model to elucidate the in vivo biology of this dsDNA animal virus.The MdSGHV genome is 124 279 bp long and has a total of 108 putative ORFs (Garcia-Maruniak et al., 2008). Comparative analysis of the genome sequences has shown that the MdSGHV is closely related to the tsetse fly virus, Glossina pallidipes SGHV (GpSGHV): both SGHVs form a monophyletic clade distinct from other circular dsDNA insect viruses (Garcia-Maruniak et al., 2009). To date, no transcriptional studies have been published on this group of viruses. Prior to initiating detailed studies on the functional aspects of ...