Study On The Machamism Of Eiav DLV Attenuated By Sited-mutation

Equine infectious anemia virus (EIAV), causes a life-long infection and chronic disease in horses characterized by periodic episodes of fever, plasma viremia, anemia, and thrombocytopenia. EIAV is a member of lentivirus subfamily of Retroviridea which related with human immunodeficiency virus. EIAV and have many resemblances on antigenicity and genome, making EIAV a wonderful animal model for HIV and research. is a member ofChinese equine infectious anemia virus donkey-leukocyte attenuated strain (EIAV-DLV) is the unique lentivirus vaccine, was developed in China in later 1970s, which is the unique one which used in large scale in the world up to now. To demonstrate the mechanism of protection of this vaccine will provide valuable references for lentivirus vaccine development.In order to investigate the molecular mechanism of the attenuation, the Chinese equine infectious anemia virus(EIAV) attenuated vaccine was developed by passaging a high virulent EIAV strain in donkey leucocytes culture in vitro for more than 110 times and completely lost pathogenicity. Here, we compared the sequences of LTR of serials passages of EIAV strain and investigation the regular mutations: the second change occured at the beginning of TAR, which changed from A to G after passage 64; the third was that the appending site of poly(A) changed from CA to AA after the 45th passage(except 55th ). This result indicated that the mutations may play important roles in virulence loss and immunogenicity increase. According to analysis results occurred in the passages during the equine infectious anemia virus (EIAV) vaccine attenuation, a full-length-gene chimeric clone, pLGFD-M, was constructed successfully at a backbone of clone pLGFD3-8 by the site-directed mutation., which the G is mutated the A at the beginning of TAR, the A is mutated the C on the appending site of poly(A). The pLGFD-M was used to transfect fetal donkey dermal (FDD) cells and passaged in FDD culture. The cell cultures were monitored by provirus DNA PCR, RT-PCR, reverse transcriptase activity assay and real-time RT-PCR, The results of RT activity and DNA PCR, RT-PCR were positive in the supernatant of cell cultures and viral particles were also clearly observed under electron microscope. The replicative ability of the chimeric viruses pLGFD-M is higher its parental virus pLGFD3-8. The pLGFD-M chimeric clone was similar to its parental virus pLGFD3-8 on replicative capability and cell tropism.There is a new important find that EIAV 5'LTR R-U5 is prior to 3'LTR is coming into cDNA 3'LTR,while it replicate and product the proteins .It is the most important that the results is investigate the replicative and attenuation...