Construction and in vitro evaluation of an infectious clone of the equine infectious anemia virus vaccine strain EIAV(FDDV) with four reverse-mutated vaccine-specific sites in the S2 gene / 病毒学报

To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecular clone pFDDVS2r1-3-4-5 was used to transfect fetal donkey dermal (FDD) cells for rescuing the derived virus vpFDDVS2r1-3-4-5. The production and replication of vpFDDVS2r1-3-4-5 in FDD cells were proved by RT-PCR, immune fluorescence assay and reverse transcriptase activity assay. Typical virons of EIAV were clearly observed under the electron microscopy. The parallel analysis of the dynamic replication of the reverse-mutated viral clone vpFDDVS2r1-3-4-5 and its parental virus vpFDDV3-8 showed that the virus with four reverse mutations in the S2 replicated only slightly slower than its parental vaccine strain in FDD cells. This result implicates that the mutations in the S2 of the EIAV vaccine strains did not significantly alter the viral replication in vitro. Further studies on the in vivo replication of the reverse-mutated viral clone are required for understanding the relationship between the S2 and the attenuated pathogenesis of EIAV attenuated vaccines.

Expression and immunogenicity of equine infectious anemia virus membrane protein GP90 / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Membrane protein GP90 of China equine infectious anemia virus (EIAV) vaccine strain (DLV) and its parental wild type LN strain were expressed with Bac-to-Bac baculovirus expression system and BALB/c mice were inoculated with purified protein, thereby to explore the availability of protein for differential diagnosis and potential for preparing genetically engineered vaccine.</p><p><b>METHODS</b>The authors infected donkey PBMC culture with China EIAV vaccine strain (DLV) and its parental wild type LN strain, extracted its proviral DNA as template, amplified the GP90 of DLV and LN, respectively, and expressed with Bac-to-Bac baculovirus expression system. The sf9 insect cells were infected with the recombinant baculovirus and the expressed proteins were purified by IMAC. BALB/c mice were inoculated with purified protein. The specific binding Abs generated in the immunized mice were determined by ELISA method. The neutralizing assay was set up to determine the neutralizing capability of the antigens generated in immunized animals.</p><p><b>RESULTS</b>The recombinant virus expressing viral antigens was determined by Western blot. The expressed proteins were purified by IMAC resulting in the protein purity of 87%(DLV) and 82%(LN), respectively. The antibody titer of the groups with and without adjuvant was 1 600 and 800, respectively. Serial 2 fold dilutions of the immunized mice sera were reacted with 100 TCID50 of EIAV. The end point of immunization assay was to protect 50% cells form CPE caused by EIAV in donkey skin cells. The neutralizing antibody titer was in the range 40 to 80 from animal immunized with and without adjuvant.</p><p><b>CONCLUSIONS</b>Membrane proteins of EIAV vaccine strain and wild type strain were successfully expressed in eukaryotic cell expression system according to the scheduled plan. The proteins showed strong immunogenicity and could activate animals to produce anti-EIAV specific antibody including neutralizing antibody to EIAV.</p>