Cowley Jeff A, Dimmock Christine M, Walker Peter J
Cooperative Research Centre for Aquaculture, CSIRO Livestock Industries, Long Pocket Laboratories, 120 Meiers Road, Indooroopilly 4068, Australia1.
J Gen Virol. 2002 Apr;83(Pt 4):927-935. doi: 10.1099/0022-1317-83-4-927.
Sequence analysis of the approximately 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 approximately 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 approximately 6 kb and approximately 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.
对斑节对虾鳃相关病毒（GAV）的单链RNA基因组约20 kb的5'末端部分进行序列分析，此前已确定其含有一个与尼多病毒目冠状病毒和动脉炎病毒成员等效的ORF1a-1b复制酶基因。对ORF1a-1b下游至3'-聚腺苷酸尾的GAV基因组剩余约6.2 kb进行序列分析，确定了两个高度保守的基因间隔序列，其中29/32个核苷酸是保守的。使用针对四个假定的GAV开放阅读框以及总RNA或聚腺苷酸选择RNA的探针进行Northern杂交，鉴定出两个3'共末端的亚基因组（sg）mRNA，大小分别约为6 kb和约5.5 kb。引物延伸和5'-RACE分析表明，亚基因组mRNA在两个保守基因间隔序列的中心区域的相同5'-AC位置起始。两种方法均未提供任何证据表明GAV亚基因组mRNA与脊椎动物冠状病毒和动脉炎病毒一样与基因组5'-前导RNA序列融合。通过对GAV感染的对虾组织总RNA进行核糖核酸酶/脱氧核糖核酸酶消化，还鉴定出了大小与26.2 kb基因组RNA和两个亚基因组RNA相当的细胞内双链（ds）RNA。仅鉴定出两个在保守基因间隔序列中相同位置起始的亚基因组mRNA，且这些亚基因组mRNA不存在融合的5'-基因组前导序列，这证实GAV的基因很少，并表明其利用的转录机制可能与脊椎动物环曲病毒相似，但与冠状病毒和动脉炎病毒不同。
