Study On Variation Of Genome And Characteristics Of Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV), a member of lentivirus family which relatedwith human immunodeficiency virus(HIV), causes a life-long infection and chronicdisease in horses characterized by periodic episodes of fever, plasma viremia, anemia,and thrombocytopenia. An attenuated EIAV vaccine had been successfully developed inthe 1970s by two strategies: first, a horse virulent Liaoning strain, EIAV-L, was passagedin donkeys in vivo for more than 113 times to get a highly virulent donkey-adapted strain(EIAV-DA); Second, EIAV-DA was extensively passaged in donkey monocyte-derivedmacrophages (MDMs) for more than 125 generations to produce the vaccine strain(EIAV-DLA), which has been shown to provide good protection to horses and donkeysagainst challenges with EIAV homology and heterology wild-type highly virulent strains.Thus, the attenuation of Chinese EIAV could be served as a valuable reference process indeveloping lentivirus vaccine, especially for HIV study. However, characteristics ofEIAV-L and EIAV-DLA need to be well determined. For that purpose, several ponieswere inoculated experimentally with EIAV-DLA, vOK8266, vOK8266chltr or nothing,respectively, and challenged with EIAV-L subsequently. Briefly, vOK8266 is a virusderived from an infectious molecular clone (designated as pOK8266) of EIAV-DLA, andvOK8266chltr is another virus derived from a chimeric infectious molecular clone(designated as pOK8266chltr) which was constructed by replaced the 5' and 3' longterminal repeats (LTR) of pOK8266 with the counterpart sequences from EIAV-L. Pre-and post-challenge, the replication dynamic of each kind of virus in vivo and theevolution of hosts antibody responses against viral gp45, p15, p26, p11 and p9 proteinswere recored. In addition, the relationship between genotype and phenotype of the twomost important viral genes, long terminal repeats and gp90, was explored. The mostresearch works were as following:Animal disease profile during immunization and challengeSeven EIAV seronegative ponies, designated as pony 1, 2, 4, 5, 6, 7 and 8, respectively,were applied in this study. Pony 4 and 5 were inoculated with vOK8266, pony 6 and 7were with vOK8266chltr, and pony 8 was with EIAV-DLA. Six-month later, all animalsincluding these five vaccinated ponies and another two seronegative ponies namely pony1 and 2 served unvaccinated controls were challenged with wild-type virulent EIAV-L.Rectal temperatures and clinical signs were recorded daily pre-and post-challenge. Nofever episode was observed within six months pre-challenge, indicating that all virusesused as vaccine were safe for animals, despite that the LTRs of vOK8266chltr were from virulent EIAV-L. Post-challenge, pony 1, 2 and 7 turned out to be disease progressorsdied after cycles of febrile episode, while the others remained health through out thethirteen-month observational period.Virus replication dynamic in vivoTo find out the replication dynamic of each kind of virus during infection in vivo, serumviral RNA loads and PBMC proviral DNA loads were detected termly pre- andpost-challenge. Standard curves used for realtime PCR and realtime RT-PCR wereestablished in this study. At most time pre-challenge, RNA loads of vOK8266chltr rangedfrom 10~4 to 10~5 copies/ml serum, which was moderately higher than thecontemporaneous loads of vOK8266 and EIAV-DLA, most of which were lower than 10~4copies/ml. This result indicates that LTR from EIAV-L has more promoter activity thanLTR form EIAV-DLA in vivo. However, similar correlation was not presented in PBMCproviral DNA loads. After challenge, disease progressor ponies were always associatedwith much high levels of serum viral RNA burdens during febrile episodes (above 10~6copies/ml) and steady stage of disease (above 10~5 copies/ml), comparing withnonprogressors. The result shows that high level of virus reproduce promotes diseaseprogression. About three months after challenge, viral RNA in serum samples from mostEIA nonprogressors initiated to be non-detactable, demonstrating that viral replicationwas almost controlled in these animals. Yet, proviral DNA was constantly detectable inPBMC samples from both EIA progressors and nonprogressors, and either pre- andpost-challenge. Role of the persistent proviral DNA in vivo for the persistent infection ofEIAV needs to be determined.Antibody responseTo evaluate the host's immuno-response against viruses used as vaccine or challengestrain, viral gp45-, p15-, p26-, p11- and p9-specific antibodies from all animals weredetected termly pre- and post-challenge, using ELISA method. After vaccination,antibody responses against viral gp45, p15 and p26 proteins in pony 4 and 5 werequicker and/or stronger than those of other ponies. Higher reproduce levels ofvOK8266chltr than those of vok8266 and EIAV-DLA should be response for such afinding. After challenge, gp45-, p15-, p26-specific antibodies from EIA progressorponies reached peaks quickly and maintained high levels since. Pony 2 was an exceptionfor it died before emergence of most antibodies. Dissimilarly, levels of these antibodiesfrom all nonprogressors except pony 6 were moderate post-challenge. Pony 6 showed asimilar antibody response to pony 7, a progressor, pre- and post-challenge, which mightbe due to the vaccination and challenge of these two ponies were in parallel. Based on allfindings above, the correlation between high levels of antibody and protection of animals was lack. Detectable p11- and p9-specific antiboies were only from EIA progressorssince challenge, implying increase of these two kinds of antibodies could be a mark ofprogressive disease.In vitro and in vivo evolutionary pattern of proviral gp90Viral gp90 gene is one of the two most variable regions of EIAV genome, the other isLTRs, both are important for virus pathogenicity. Knowledge of evolutionary patterns ofthese two genes is necessary to develop strategy to prevent the epidemic EIAV. TotalDNA isolated from PBMCs collected termly from pony 1, 4 (or 5), 7 and 8 pre- andpost-challenge were used as template in nested PCRs for mutiple amplification offragments encompassing proviral gp90 hypervariable regions 2 (V2) to 5 (V5). The aasequences obtained from ponies during vaccine period demonstrated that the gp90 ofEIAV-DLA, being a quasispecies, changed to be more heterologous in vivo. NewEIAV-DLA sequences with mutations toward EIAV-L were observed, suggesting risingrisk of increasing virulence. Similar cases were also found in sequences from poniesinoculated with vOK8266 and vOK8266chltr. After animals were infected with EIAV-L,only sequences representing EIAV-L could be obtained, and high levels of variation wereonly observed in the V3 region in sequences from EIA progressor ponies, while onlymoderate levels were in sequences from nonprogressors. New ferbile episodes werealways associated with new quasispecies emerging by PND mutation, revealing potentialrole of the PND mutaion for the escape of virus from immuno-response, whichcontribute to persistent infection of EIAV and promote disease progression. The ds/dnvalue for sequences indicated selection of immune responses was positive in progressorsafter acute stage but weak in nonprogressors post-challenge. In addition, high number ofdefective variants obtained only from nonprogressors, which may correlate with thestep-down dynamic virus replication and promote disease survival. In-frame stop codonswere frequently localized in a defect "hot spot" in the V4 region.In vitro and in vivo evolutionary pattern of proviral LTRsProviral LTR genes were amplified by mutiple seminested PCRs from MDM culturesexperimentally infected by EIAV-DA and EIAV-DLA, PBMCs with EIAV-L proviralgenomes inside, and PBMCs obtained termly from pony 1, 4 and 8 pre- and/orpost-challenge. The 11, 10 and 14 LTR sequences from EIAV-L, EIAV-DA andEIAV-DLA, respectively, demonstrated that the homologous population of EIAV-L andEIAV-DA converted to be a heterologous quasispecies of EIAV-DLA, revealing that thein vitro passages of EIAV attenuation process would mainly account for the variableLTRs residing in EIAV-DLA. Two hypervariable regions were defined in LTR U3 and Rregions of EIAV-DLA, respectively. Interestingly, experimental inoculation of EIAV-DLA into pony 8 resulted in a restriction of the LTR variation, and LTR-DLA12analogs were selected as the predominant type. Sequences from pony 1 and 4 showedthat LTRs were extremely conservative in vivo.Assay of LTR promoter activity in vitroRole of mutations in the hypervariable regions A and B defined in LTR-DLA needs tobe determined. Several representative LTR sequences were selected from EIAV-L,EIAV-DA and EIAV-DLA, respectively, for constructing LTR/CAT contexts. Twochimeric pLTR/CAT plasmids were generated by replacing the R region of LTR fromEIAV-L with the counterparts of two LTR sequences from EIAV-DLA. Equine MDMcultures were transfected with pLTR/CAT plasimds in the presence or absence of EIAV-LTat protein. The result, the basal activities of all LTR variants were quite low, and thedifferences among them were non-significant. LTRs from EIAV-DLA showed higher Tattransactivated activity than LTRs from virulent strains. By using chimeric clones betweenwild-type EIAV-LLTR and vaccine LTR, the main difference of activity was mapped tothe changes of R region, rather than U3 region. In particular, the A to G mutation fromLTR-L to LTR-DLA at the beginning of TAR could affect secondary structure of TAR,perhaps influencing Tat-TAR interaction. In addition, the considerable changes at U3-Rborder may be critical for viral RNA synthesis.