| Astrovirus are known to infect mammals,such as human,pigs,bats,dogs,cats and seseveral avian species,such as chickens,turkeys,pigeons,and are causative agents of mostly enteric diseases.Duck astrovirus(DAst V)is the member of the Avian astroviruses belonging to the astroviridae family and is non-enveloped,positive-sense and single-stranded RNA viruse.Duck astrovirus can infect young duck mainly within the 3-week-old ducklings.Unlike other species,astroviruses in ducks have been associated with a fatal hepatitis,characterized by liver bleeding,convulsions and neurological symptoms such as opisthotonos.In recent years,the study found that DAst V and DHAV coinfected frequently,which further increased the difficulty of DHV prevention and control.In order to have a thorough understanding of the virus genome structure and function,genome replication and transcription regulation mechanism,in this study,an infectious full-length cDNA clone of DAst V-1 D51 strain was constructed and we studied its propagation in BHK-21 cells.And a SYBR Green I Real-time Quantitative PCR method was established for the distribution of the rescued virus in the duck body to do the research.1.The prokaryotic expression of ORF2 protein and the preparation of serumTo the success of the IFA test,duck astrovirus serum was prepared.Three fragments of the ORF2 gene were amplified and cloned into pET-32 a vector,respectively,resulting in expression vectors.The recombinant proteins were expressed in Escherichia coli BL21 and purified.Approximately 100?g of three proteins was mixed with Freund's complete adjuvant and subcutaneously inoculated into BALB/C mice.Two further inoculations of the proteins into Freund's incomplete adjuvant were done at 14-day intervals.Serum were collected 10 days after the third inoculation.2.Construction of a DNA-launched infectious cDNA clone of a duck astrovirus type 1D51 strain and virus rescueAccording to the vector pABX,ribozyme sequence and viral genome sequence of restriction maps,four pairs of oligonucleotides were designed based on the full length genomic sequence of DAst V-1 D51 strain,which has completed genome sequencing in ourlaboratory.Eleven overlapping fragments of the DAst V-1 D51 strain genome with overlap-extension PCR were fused into four long fragments(FA,FB,FC,FD).Ribozyme sequences were introducted at both termini of the viral genomic cDNA by primers and the single restriction site of Xho I was added to the 3'end by the process of primer design.FA,FB were cloned into pEASY-TI vector respectively(named plasmid A,plasmid B),while the FC,FD was cloned into pMD18-T vector(named plamid C,plasmid D).The plasmid A and the plasmid B were digested with KpnI and BamHI,and ligated with T4 DNAligase,producing plasmid AB in the vector pEASY-T1.The plasmid AB was digested with Asc I and BamHI and cloned into the vector pABX at the same restriction site.Subsequently,the fragment C which was digested with EcoRV and BamHI was inserted into the backbone vector pABX-AB by double-digested with EcoRV and BamHI site(resulting in pABX-ABC).Lastly,the full-length genomic cDNA of DAst V(pABX-DAst V)was generated by re-ligating the fragment D and the vetor pABX-ABC which were digested with unique restriction enzyme recognition sites BglII and Xho I.In addition,the BglII recognition site without causing amino acid substitution was created by a silent mutation(nucleotide position 6234,from C to T)to distinguish it from parental virus.The complete genome sequence of recombination plasmid DNA was sequenced and compared with the parental virus.The result shows that a nucleotides change during the cDNA cloning procedures in the full-length cDNA.In addition,a nucleotide change of C to T at position 6234 was incorporated artificially.The recombination plasmid DNA was used to transfect BHK-21 cells.As a negative control,Lipofectamine2000 Transfenction Reagent with PBS was added to BHK-21 cells.The identification results by RT-PCR and IFA were regarded as positive for DAst V-1.After the qualification experiment on genetic marker,we are so sure we have obtained rescued virus.The success on successful construction of full-length cDNA clone of DAst V-1 D51 strain provides a useful tool for future research on genomic functions and molecular pathogenesis of DAst V-1.3.The study of propagation of the virus in the presence of trypsinBecause there is no suitable cell line proliferation of the astrovirus,the study of the virus also has some limitations.Some researches said that human astrovirus can propagate in the presence of trypsin.So we studied the method of propagation of the virus in the presence oftrypsin.The virus was obtained from transfected cells after three times freezing-thawes.Prior to infection,the virus samples were activated with 200?g/mL trypsin for 1 hour at 37?.Before adsorption,the monolayers were washed twice to remove any residual serum.The virus samples together with 400?g/mL soybean trypsin inhibitor(Sigma)were added to the cells.After a 1h adsorption period,2mL DMEM containing 3?g/mL trypsin were added and then incubated for 24 h.In the presence of trypsin the virus was passed 6 times as described above.4.Construction and application of SYBR Green I Real-time Quantitative PCR methodThe standard curve for DAst V-1 was generated by the serial 10-fold dilutions of the standard linear templates,range from 7.3×109~7.3×102 gene copies per reaction.The linear correlation(R2)value was 0.998,and the reaction efficiencies was 94.4 %.The cDNA copy numbers of DAst V-1 for unknown samples were quantified using the equations YDAst V-1 =-3.4811X+38.429(Y=threshold cycle,X =log starting quantity).Specificity,sensitivity and reproducibility of the SYBR Green-based real-time PCR assay were tested.The two-day duckings were injected with 1mL rescued virus,heart,liver,spleen,kidney,thymus,mursa of fabricius,intestine were tested with real-time quantitative PCR,found that the rescued virus can be detected in every organs,showed that the rescued virus can proliferate in the duck body. |
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