Analysis On Genomic Organization Of Ectropis Oblique Picorna-Like Virus

Evtropis oblique is a major pest in tea field. An icosahedral virus was first identified from dead larvae of the tea looper (Ectropis obliqua) caused by a granulosis virus (GV) infection in 2000. Using differential and sucrose gradient centrifugation, we previously purified a non-enveloped, isometric ssRNA virus (26 nm in diameter) from GV-infected larvae of Ectropis obliqua. The virus was named Ectropis obliqua picorna-like virus (EoPV) .Electron microscopic observation of purified virions were non-enveloped, isometric particles with a diameter of 26nm. Virions contain a about 9.4kb polyadenylated, ssRNA .Two major capsid proteins (31.5 and 28.8kD) were resoved by 16% SDS-PAGE, and the amount of the later is 2.5 times more than that of the former.To identify the taxonomy position of EoPV, a serial overlap clones were constructed by walking primers, RT-PCR and 5'RACE techniques. The complete genomic sequence was obtained by determining all these clones. The genomic RNA of EoPV is 9394 nt in length (GeneBank No. AY365024) . The sequence is A/T rich (54.6%) , which is agreement with picornavirus. EoPV genome contains a single, large open reading frame (nt 391-9351) encoding a polyprotein of 2987 aa.By analyzing the 5'UTR of EoPV , it was found that like the 5'UTRs of mammal picornaviruses, the 5'UTR of EoPV was rich in A/T, and had many AUG and small cistrons up stream the authentic initiation codon. The second structure of 5'UTR of EoPV was predicted with mfold software. There were 4 stem-loops, and conserved motifs of mammal IRESs, including stem-loop A (contains conserved motif,GNRA) , loop B (A / C rich) and polypyrimidine tract. Based on the characteristics of 5'UTR of EoPV, the genome was suggested to be translated with IRES mechanism. Compare with the other 14 insects' picornaviruses downloaded from Genebank, 5' UTR of EoPV was shorter than that of the other insects and mammal picornaviruses. The EoPV 3'UTR (43 nt) was similar in size to that of mammalian picornaviruses (40-120 nt) and did not have a typical polyadenylation signal (AAUAAA) , found in many eukaryotic mRNAs.The genome of EoPV contains a single, large open reading frame (nt 391-9351) encoding a polyprotein of 2987 aa. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, protease andRNA-dependent RNA polymerase proteins are found on the genome in order in the 5'— 3' direction. All structural genes were located at the 5' terminus. In terms of sequence similarity, identity and genome organization, EoPV resembles mammalian picornaviruses and three other insect picorna-like viruses: Infectious flacherie virus of silkworm, Sacbrood virus ofhoneybee and Perina nuda picorna-like virus (PnPV) . Phylogenetic analysis showed that EoPV is most closely related to PnPV and suggests that these four insect picorna-like viruses might constitute a new group of insect-infectious RNA viruses, iflavirus.