Cloning And Sequence Analyzing Of The Whole Genome Of EIAV L Provirus

Equine infectious anemia virus(EIAV) is the etiological agent of equine infectious anemia(EIA). EIAV and human immunodeficiency virus(HIV) are both members of lentivirus of retrovirus family and related in morphology, genome organization, heredity, antigenicity, cell tropism, variability and so on. EIAV can be served as an animal model for studying the pathogenic mechanism of HIV. Equine infectious anemia virus donkey leukocyte attenuated vaccine(DLA-IAV) developed in China was the only successful lent ivirus vaccine in the world, and has been applied extensively in China against EIA for twenty years. The vaccine was developed by first passaging EIAV L strain(EIAV L) in donkeys and donkeydapted equine infectious anemia virus(DA-IAV) was obtained, then attenuating DAIAV in donkey leukocytes. To elucidate the molecular basis of the virulence attenuation of DLAJAV and lay theoretical foundation for designing HIV vaccine, the whole genome EIAV L provirus was cloned and sequenced on the basis of the complete sequence of DAIAV and DLAIAV.In this study, peripheral blood lymphocytes (PBL) were collected from a horse infected with EIAV L.The PBL DNAs were extracted and used as temple. The EIAV L proviral DNA was amplified by polymerase chain reaction(PCR) in four parts, which covered the whole proviral DNA genome. Each of the four parts was cloned into the plasmid vector pBluescript SK. Through subclone, the recombinant plasmid pLY containing the whole genome of EIAV L provirus was finally obtained and sequenced. The genome of EIAV L was determined to be 8235 bp in length, and G+C content was 38%. Both ends of EIAV L provirus are identical long terminal repeat sequence(LTR) of 3l6bp in length. The LTR include U3,R,and U5 regions in turn, and the sizes of them are l97bp, 8Obp and 39 bp , respectively. The genome of EIAV L provirus contained three long open reading frames(ORFs) corresponding to gag, po] and env genes , respectively. Gag gene is l200bp located at 613?912. Pol gene is 3402bp at position 1708?109. Env gene is 2592bp at position 5305?896. The provirus has three small ORFs,i.e.Sl,S2 and S3,with sizes of 153bp(nucleotides5ll3?265), 204bp (nucleotides 5279?482) and 402bp (nucleotides7245?646), respectively.The DNasis analyzing results showed that the nucleotide sequence homology between EIAV L and DAIAV is 98. 4%, and 96. 9% between EIAV L arid DLAIAV. The high homology between these strains shows their relative. This is consistent with the derivation process of DLA桬IAV.There is high difference in nucleotide sequence between EIAV L and other alien EIAV strains, and the homology is only about 79%. The homology of Rlase and Gag protein amino acid between EIAV L andother alien strains are 86% and 79% , respectively. These results show that there is a distant relationship between EIAV L and other alien strains.Although there exists high differences among EJAV L, DL桬IAV, DLA桬IAV and alien EIAV strains, but several known domains which have important function in EIAV life cycle are reserved . YXXL motif in P9, the two zinc finger in P11, D桾桮 motif near amino terminal and L/I桮桾桪/N motif near carboxyl terminal in protease are identical in all EIAV strains. There are six conserved N條inked glycosylation sites in Gp90 and four in Gp45 among all EIAV strains. The two cysteine residue, which function in keeping Gp45 constellation, the corn domain and glutamic acid in Tat protein and the basic domain in Rev protein are conserved in all EIAV strains. The determination of these conserved domains provided valuable information for further study of EIAV.There is no start cordon桝TG within gag梡ol overlapping zone in EIAV L, DA桬IAV and DLA桬IAV. The result shows that there is a conserved stem條oop structure by analyzing this zone of all EIAV strains. This further confirmed the translation mechanism of poi gene is ribosomal frame shifting.TAR is the effect region of the trans梐ctivation protein桾at. Through comparison of TAR between EIAV L and other strains, variation in the origination site of T...