Gill-associated nidovirus of Penaeus monodon prawns transcribes 3′-coterminal subgenomic mRNAs that do not possess 5′-leader sequences

Abstract

Sequence analysis of the ∼20 kb 5′-terminal portion of the ssRNA genome of gill-associated virus (GAV) of Penaeus monodon prawns has previously established that it contains an ORF1a–1b replicase gene equivalent to those of the coronavirus and arterivirus members of the order Nidovirales. Sequence analysis of the remaining ∼6·2 kb of the GAV genome downstream of ORF1a–1b to a 3′-poly(A) tail has identified two highly conserved intergenic sequences in which 29/32 nucleotides are conserved. Northern hybridization using probes to the four putative GAV ORFs and either total or poly(A)-selected RNA identified two 3′-coterminal subgenomic (sg) mRNAs of ∼6 kb and ∼5·5 kb. Primer extension and 5′-RACE analyses showed that the sgmRNAs initiate at the same 5′-AC positions in the central region of the two conserved intergenic sequences. Neither method provided any evidence that the GAV sgmRNAs are fused to genomic 5′-leader RNA sequences as is the case with vertebrate coronaviruses and arteriviruses. Intracellular double-stranded (ds)RNAs equivalent in size to the 26·2 kb genomic RNA and two sgRNAs were also identified by RNase/DNase digestion of total RNA from GAV-infected prawn tissue. The identification of only two sgmRNAs that initiate at the same position in conserved intergenic sequences and the absence of 5′-genomic leader sequences fused to these sgmRNAs confirms that GAV has few genes and suggests that it utilizes a transcription mechanism possibly similar to the vertebrate toroviruses but distinct from coronaviruses and arteriviruses.