Construction And Characterization Of A Chimeric Infectious Clone Of Chinese Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a member of lentivirus family which related with human immunodeficiency virus, causes a life-long infection and chronic disease in horses characterized by periodic episodes of fever, plasma viremia, anemia, and thrombocytopenia. Chinese equine infectious anemia virus donkey-leukocyte attenuated strain (EIAV-DLV) is the unique lentivirus vaccine, was developed in China in later 1970s, which has been applied extensively for over 20 years in China mainland. This vaccine strain can provide fully protection to horses and donkeys against challenge with EIAV homology and heterology wide type high virulent strains. Vaccine DLV had been proved to be safe and effective for horse and donkeys after being used in large scale in China mainland for over 70 million vaccinated horses for years. And it is very stable in that no virulent reversion has been observed in numerous tests involving several hundred horses and donkeys. DLA-EIAV can be served as the animal model for studying on human immunodeficiency virus (HIV). The vaccine was developed in two stages. (i) A carefully selected field isolate, EIAV LN, was passaged 133 times in vivo in donkeys to generate the highly virulent and pathogenic strain D510, which kills animals within a few weeks. (ii) D510 went through 135 passages in vitro to obtain vaccine strains DLV. In order to investigate the molecular mechanism of the attenuation and protection of donkey leukocyte attenuated equine infectious anemia virus (DLA-EIAV), we investigated the evolution of LTR of 24 out of 110 passages during Chinese EIAV attenuation. The EIAV enhancer region is surprisingly hypervariable. According to analysis results occurred in the passages during the equine infectious anemia virus (EIAV) vaccine attenuation, a full-length-gene chimeric clone, pLGFD9-12, was constructed successfully at a backbone of clone pLGFD3-8 by substitution with LTR U3 region of a virulent EIAV strain. The pLGFD9-12 was used to transfect fetal donkey dermal (FDD) cells and passaged in donkey leukocyte (DL) 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 pLGFD9-12 chimeric viruses and its parental virus pLGFD3-8 have no obvious differences. The level of replication of the pLGFD9-12 chimeric clone that was cultured in DL cells was higher than that in FDD cells. The pLGFD9-12 chimeric clone was similar to its parental virus pLGFD3-8 on replicative capability and cell tropism, E-box motif and GATA motif have no distinct function on viral replication and cell tropism. The more characteristics on virulence and replication ability of pLGFD9-12 will be evaluated in vivo. In this study, five EIAV-negative horses were randomly divided into three groups, one horse(2#) in Group 1 was healthy control, two horses(15# and 17#) in Group 2 were inoculated with attenuated vaccine infectious molecular clone pLGFD3-8, two horses (20# and 30#)in Group 3 were inoculated with LTR chimeric molecular clone pLGFD9-12. Animals inoculated with the following virus stocks received a virus dose of 5*105TCID50 by subcutaneous injecting. Rectal temperatures and clinical symptom of animals were monitored twice daily. Blood samples were obtained pre-and postinfection for determination of complete blood counts .Blood and serum samples were collected to quanitify virus, to monitor the development of blood routine, viral RNA load in plasma of experimental animals, the level of lymphocyte CTL function and lymphocyte proliferation function, The more characteristics on virulence and replication ability of pLGFD9-12 will be evaluated in vivo. During monitor days, all inoculated groups showed no abnomal change, horse 15# showed a transient fever after 47 day post infection, but quickly recovered. It was not typical EIAV symptom and seems not caused by EIAV. Results of analysis blood routine show the amout of WBC and HGB have no obvious changes. The RNA copies in plasma of inoculated animals were relatively low. Replicative capability of LTR chimeric clone pLGFD9-12 was similar to its parental virus pLGFD3-8 in vivo. CTL and lymphocyte proliferation assay showed all the inoculated animal have similar cellular immune responses against EIAV. These results provides an important foundation in molecular biology for further study on mechanism of replication and attenuation of the DLA-EIAV, as well as the establishment of protective immunity for EIAV, which might provide important insights into the development of other lentivirus vaccines.