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Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus

¹ Department of Entomology and Nematology, PO Box 110620, University of Florida, Gainesville, FL 32611-0620, USA
² Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, 9 Gamaa Street, Giza 12619, Egypt

Correspondence
Tamer Z. Salem
salemtz{at}ufl.edu

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 3'-ends (3'-RACE) and RT-PCR. Direct sequencing of 3'-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3'-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3'-RACE or RT-PCR. A series of 5'-RACE reactions were conducted on selected ORFs that were identified by 3'-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 3' untranslated region (3'-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 3'-RACE products without cloning served as an alternative approach to detect both 3'-UTRs and transcript variants of this large DNA virus.

Two supplementary figures, showing sequences flanking the cleavage sites of MdSGHV transcripts and the 5'-UTR sequences of selected ORFs that transcribed in tandem, and three supplementary tables, listing primers used for 3'-RACE, reverse primers used for 5'-RACE and primers used for RT-PCR, are available with the online version of this paper.