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The Establishment of a Diagnostic Method to Differentiate American Prevalent EIAV Strain from Chinese Attenuated Vaccine Strain and the Development of an EIAV Marker Vaccine

Author: GengQingHua
Tutor: XiangWenHuaï¼›ShenRongXian
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: Equine infectious anemia virus Nested multiplex PCR Differentiation diagnosis Marker vaccine
CLC: S852.5
Type: PhD thesis
Year: 2006
Downloads: 115
Quote: 1
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Abstract


The purpose of this Ph.D. research is to establish a diagnostic method to differentiate horses infected with American strains of equine infectious anemia virus (EIAV) from the population immunized with the Chinese attenuated EIAV vaccine strain. The proviral genome of an Argentina EIAV strain (Agen-EIAV) was cloned by PCR. The complete sequence of Agen-EIAV was obtained by combining the sequencing data of PCR products and referencing the published EIAV sequences. The Agen-EIAV genome was 8,227bp in length, which compares with EIAV strains of LN , DLV and AF028232, with the homology of 65%, 65% and 75%, respectively. In detail, the homology of gag, pol, env, gp90 and gp45 in Agen-EIAV was 99%, 99%, 75.6%, 69.4% and 80.4%, respectively, when compared with the corresponding gene fragments in the EIAV American strain AF028232. The homologies of amino acid sequences of Gag and gp90 between Agen-EIAV and American strains were higher than those between Agen-EIAV and Chinese EIAV strains.Since the in vivo mutation rates of certain EIAV genome regions are high, we investigated the genetic evolution of two functionally distinct regions (gag and gp90) of Agen-EIAV provirus during recurring febrile episodes. Proviral DNA from peripheral blood mononucleocytes (PBMC), which were prepared from an Agen-EIAV-infected pony during sequential febrile episodes (23, 40, 51 and 70 days postinfection), were amplified and analyzed. The variation of gag was not significant during four febrile episodes, with the percentage of nucleotide variation less than 1%. Therefore, this region can be selected for primers of differentiate diagnosis. Instead, the variance of gp90 was as high as 8-10%. The comparison of variable nucleotide showed that mutations were not randomly distributed, but clustered into four variables regions, A1-A4. Among these regions, A1, A3 and A4 were mutated only once during the four febrile episodes, while A2 was mutated at the third febrile episodes and then was recovered at the fourth episode. The study showed that the proviral status of EIAV was very steady after the EIAV genome was integrated into cellular chromosomes. This status of EIAV offers a credible basis for PCR assay of EIAV infection.Ten primers were designed based on the comparison of complete sequences of four EIAV American strains and the Chinese vaccine strain DLV. Corresponding viral genome fragments were amplified and analyzed from donkey leucocytes by PCR using these ten primers. Six primers from gag were selected to detect the differences between EIAV American strains and DLV. Nine horses were inoculated with DLV and two EIAV American strains, the Wyoming strain and the Argentina strain. Two health animals were used as negative control. The experimental conditions of nest multiplex PCR, which was applied to identify different EIAV strains using the selected six primers, were farther optimized. When primers of c1/c2n/c3n were used, two fragments, with sizes of 675bp and 400bp, respectively, were amplified from DLV-infected cells, while only one segment of 675bp was obtained from cells inoculated with American strains. When the combination of primers was c1/af2n/c3n, two fragments, with the sizes of 675bp and 400bp, were amplified from American strains, while only one segment of 675bp wasobtained from DLV-infected cells. Our results indicate that a nest multiplex PCR-based assay, which distinguishes EIAV American strains from the donkey leucocyte-attenuated vaccine strain of EIAV, is developed. This method detects minimal 102 copies of EIAV genome/ml of chromosomal DNA from EIAV-infected cells.Furthermore, two mutated EIAV infectious clones (pLG-5B19 and pLG-np2 ) were constructed from in a well characterized infectious EIAV clone, pLGFD3-8 (He, 2000). One of the mutated clones was introduced two stop codons in S2 to silent the expression of this gene (pLG-np2). The other mutated clone had an insertion of the cDNA of murine hepatitis virus glycoprotein predominant B cell episode 5B19 to substitute the first 7 amino acid of S2 protein (pLG-5B19). These two infectious clones were transfected into FDD cells, and their replication kinetics were examined. The clone pLG-5B19 showed replication kinetics similar to those of the parental virus in FDD, while the replication of clone pLG-np2 was consistently delayed compared to the replication of parental EIAV clone pLGFD3-8. However, the final viral titers of these two S2 mutants eventually reached to levels similar to those of pLGFD3-8. Large numbers of viral particles were also clearly observed under electron microscope in cells infected by both of these mutated EIAV clones.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Serological
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