The Role Of Mutations In The S2 Gene In The Attenuated Virulence Of The Chinese Attenuated Vaccine For Equine Infectious Anemia

The S2 gene of equine infectious anemia virus (EIAV) is known related to the virulence of this virus. However, the exact function of S2 is still unclear. By sequence comparison of EIAV virulent and vaccine strains, four stable mutation sites were identified in the S2 gene of Chinese EIAV attenuated vaccine strains occurred during the attenuation process. To investigate the role of these stable mutations in the attenuated virulence of EIAV vaccine strains and the function of S2, these four stable mutation sites in the S2 gene of pFDD3-8, an infectious molecular clone of the EIAV vaccine strain EIAVFDDV, were reverse-mutated. Six infectious molecular clones were constructed, which contained different combinations of reverse-mutated sites in the S2. Viruses derived from these mutated clones were rescued, and termed vpFDS2r1-3-4-5, vpFDS2r3-4, vpFDS2r1-3-4, vpFDS2r3-4-5, vpFDS2r1 and vpFDS2r5, respectively, according to the numbers and sites of the mutation. The in vitro replication properties of these 6 derived viruses were detected by quantitative real-time PCR, activity assay of reverse transcriptase and Western blot. Results revealed that except the virus vpFDDS2r3-4-5, 5 of these 6 revise-mutated viruses replicated significantly slower than the parental viral clone vpFDD3-8 in fetal donkey dermal (FDD) cells (p<0.01). In contract, only the virus vpFDDS2r1-3-4-5, which had all the four sites reverse-mutated, demonstrated a significantly delayed dynamic replication rate in equine monocyte-derived macrophages (EMDM) when compared with vpFDD3-8 (p<0.05). These results implicate that mutations in the S2 of EIAV vaccine strains facilitate the replication of these in vitro-attenuated viruses in FDD cells. However, the synergetic effect of all the four stable-mutated sites in the S2 protein of the EIAV vaccine strains may required for better growth of these virus in EMDM. Since EMDM is the major target cell for EIAV in vivo, the viral clone vpFDDS2r1-3-4-5 was applied for further studies on the features of EIAV vaccine strain with reverse-mutated S2.To evaluate the function of the reverse-mutated S2 gene in vivo, the pathogenicity of the viral clone vpFDS2r1-3-4-5, which was reverse-mutated at all the 4 mutation sites of the S2, was evaluated by infecting horses and comparing with the parental vaccine infectious clone vpFDD3-8. Twelve EIAV-negative horses were randomly divided into three groups, four horses for each group, and were inoculated with vpFDD3-8 (Group 1), vpFDS2r1-3-4-5 (Group 2), and saline (Group3, health control), respectively. The body temperature, platelet number and plasma viral load were regularly measured for horses of all the three groups. Results demonstrated that horses infected with vpFDS2r1-3-4-5 appeared typical signs of EIA, i.e. the increases in body temperature and plasma viral load and the decrease in platelet number at certain days post infection. Furthermore, statistic analysis revealed that the plasma viral loads in animals of the Group 2 were significantly higher than that of the Group 1 (p<0.01), especially during 23-46 days post infection (DPI). However, these symptoms of EIA were mild, and were below the standard for acute EIA episodes.In conclusion, the above studies indicate that firstly the S2 gene is important for EIAV replication in vivo. However, other genes may also be involved. Secondly, the S2 is one of the major genes that determine the pathogenicity of the EIAV virulent strains through regulating the replication efficiency. Thirdly, although reverse-mutations in the S2 of EIAV vaccine strain increased the replication level in the host, the reverse-mutated virus was nonpathogenic. This fact indicates that reverse-mutations in single gene of the EIAV vaccine does not alter the attenuated virulence, which provides further evidence for the safety of the Chinese attenuated vaccine.