| Equine infectious anemia virus (EIAV) is a member of the lentivirus subfamily of retroviruses and causes a persistent infection and chronic disease in horses worldwide. The same as the other lentivirus, under the immune pressure, EIAV gene tend to variation. The variant can escape the immune response built by organism, lead to persistent infection of the host animal. EIAV is similar to HIV-1about some characters that include high mutation rate and immune escape mechanism, which make EIAV as the model to research molecular pathogenesis mechanism and immunologic mechanism of HIV-1and other lentivirus.Despite the worldwide prevalence of EIAV infections, experimental studies to date have centered on relatively few viral isolates. analyses of viral pathogenesis have essentially focused on a strain of EIAV termed Wyoming (isolated in North America) and its derivatives (EIAVpv、EIAVUK and EIAVwsus) while a minority of reported studies have utilized a Chinese strain EIAVLN40and its derivatives (EIAVDLV and EIAVFDDV) and a Japanese strain V70and its derivatives V26. The majority of research in genetic variation focus on between strains, but in vivo evolutionary analysis of each strain mainly concentrate on EIAV gp90gene, but the variation of gag gene and gp45gene has not been investigated in vivo.To study the evolution rule of the EIAVLN40p15, p9, gp45-Ⅰ and gp45-Ⅱ in vivo,3horses were inoculated with EIAVLN401×105TCID5o respectively, body temperature, platelet and viral copies of these horses were monitored at regular intervals, the peripheral blood lymphocytes(PBL) was collected from blood at different time points. DNA extracted from PBL was used as the template strand to amplify the EIAV proviral DNA of p15, p9and gp45region in nested PCR technique. cloning and identification were conducted and sequencing were analyzed through several bioinformatics software.The results showed that all the horses didn’t appear typical signs of equine infectious anemia after inoculation. The number of viral copies were below1×106copies/ml, simultaneously body temperature and platelets were within normal limits. EIAVLN40p15、 p9、gp45-Ⅰand gp45-Ⅱ gene had a lot of mutations in experimentally infected horses, which caused substitutions of translated amino acid residues in EIAV replicated in the hosts. substitutions of translated amino acid residues of p15gene were in the16thN/S and122nd M/E about three horses; substitutions of translated amino acid residues of p9gene were in the67th F/L、115th E/G and122nd K/M about three horses; substitutions of translated amino acid residues of gp45-Ⅰ gene were in the ninth T/F/L and about three horses; substitutions of translated amino acid residues of gp45-Ⅱ gene were in the21st E/K、29thG/S'33rdV/T about three horses. In addition, the p15、p9、gp45-Ⅰ and gp45-Ⅱgene sequences of samples at the2nd period from horse#2and samples at the1st period from horse#3appeared lower divergence to the consensus p15、p9、gp45-Ⅰand gp45-Ⅱ gene sequence of EIAV pathogenic strains EIAVLN40.The result showed that the average divergence of nucleotide and amino acid of p15gene were1.8%(0-2.9%) and1.7%(0-3.6%) respectively,p9gene were2.5%(0.3%-3.8%) and2.9%(0-4.8%) respectively, gp45-Ⅰ gene were1.3%(0.3-2.1%) and1.8%(0-3.2%) respectively, gp45-Ⅱ gene were2.1%(0-3.4%) and2.6%(0-4.7%) respectively, compared with EIAVLN40.Moreover, during the fever period of EIA, the divergence of each nucleotide and amino acid was in a lower level or even no change; while the rate was relatively high in the sub-clinical stage. phylogenetic tree analysis of p15gene (the#2horse at68day and#3horse at22day post-infection) showed that the gene sequences in fever period were on the same branch with EIAVLN40, but others at other time points were on another branch; evolutionary analysis of other genes is similar to the p15gene.This study first analyze evolution in vivo of EIAVLN40p15、p9、gp45-Ⅰand gp45-Ⅱ gene, which will facilitate the studies on procedures of infection of pathogenic EIAVLN40and attenuated mechanism. |
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