Molecular Cloning The Genes for Waterfowl Parvoviral Proteins and Characterization of Their Antigenicity
Parvoviruses cause dreadful enteritis in waterfowls and lead to tremendous financial losses. This study aims at developing effective way to prevent waterfowl parvoviral infection. Duck parvoviruses (DPVs) and goose parvoviruses (GPVs) were isolated from organs of infected waterfowls. The presence of virus in the specimens was identified using polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis. To reveal the genetic variation of viral capsid proteins (VPs), full length VPs gene were amplified and sequenced. The sequence data indicated the sequences diverge 4.1 to 4.4% among viral strains isolated during 1990 to 1999. The variant amino acids cluster in the common regions of VP3 at residues 203-266 and 482-534, which overlaps with the regions proposed to expose on the outer surfaces of parvoviral particles. These data implying that selective pressure from host immune system might play a part. The nucleotide sequences of VPs also reveal that DPV and GPV share 77 % similarity at the DNA, and 84.6% at the protein level. The most variable regions reside in the N-terminal of VP2 before the initiation codon of VP3 with 35% (19/54) amino acids divergence. This study also reveals the presence of conserved strain-specific residues in VPs and these residues seldom vary among different isolates of the same virus, suggesting that they might be important in maintaining viral structure or host specificity which worth further investigation. To investigate the antigenicity of VPs, the GPV genomic DNA encoding common region of VPs was fused in frame with glutathione S-transferase (GST) gene for the expression of GST-GPV (248-516) fusion protein in bacterial cells. Purified fusion protein was used as immunogen for the generation of rabbit anti-GPV (248-516) antiserum. The potential diagnostic usage was confirmed by the fact that this antiserum was able to differentiate between viral infected and uninfected primary embryonic fibroblast cells by immunocytochemical analysis.
In addition, VPs in purified DPV and GPV virions were analyzed by Western blotting. This antiserum detected two prominent proteins bands with the molecule weight of 80 and 70 kilodaltons, which correspond to the sizes of VP1 and VP2 reported in the literature. The fact that VP1 of DPV reacts weakly with this antiserum suggests the existence of antigenic discrepancy between DPV and GPV. For the purpose of developing subunit vaccine for the control of Derzy's disease, recombinant full length VPs were expressed using both prokaryotic, GST and histidine-tagged fusion proteins, and eukaryotic, baculovirus and mammalian vero cell, expression systems. After large- scale production and purification, same amount of 4 recombinant VPs were individually used to immunize 1-week-old geese. The antibodies induced after immunization were then evaluated by enzyme-linked immunosorbent assay (ELISA). All four recombinant proteins stimulate approximately 7 to 8 folds increases of ELISA antibodies titers, and together with
preliminary data of safety tests suggest a potential usage as subunit vaccine for the control of parvoviral infection.
Advisor:Jiin-Tsuey Cheng; Chau-Liang Wu; Ai-Li Shiau; Mao-Yion Lin; Ming-Jeng Pan
School:National Sun Yat-Sen University
School Location:China - Taiwan
Source Type:Master's Thesis
Keywords:waterfowl parvovirus capsid protein antigenicity genetic variation
Date of Publication:08/31/2001