Identification Of Novel Duck Parvovirus DS15 Strain And Expression Of VP2 Protein In Insect Cells

III Since 2014,an infectious disease has emerged in commercial broiler duck breeding farm in eastern China.The diseased ducks displayed symptoms such as short beak with protruding tongues and significant growth retardation.Based on the Clinical signs,the disease was tentatively designated short break and dwarfism syndrome(SBDS).The morbidity among ducks has been 20% to 30%.SBDS has caused significant economic losses in the waterfowl industry.The pathogen of the new infectious disease is not clear.Effective vaccine and therapeutic drugs were lacking for this disease.In this study,a novel duck parvovirus was isolated from diseased ducks in Dangshan,Anhui province.The genome of the pathogen was cloned and sequenced,and then we used baculovirus-insect cell expression system to expression structural proteins of the pathogen and evaluate the immunogenicity.This laid a good foundation of developing subunit vaccine.In this study,we first analyzed tissues from affected duck and preliminary detected it was a novel duck parvovirus after other common duck virus were excluded.The supernatant of splanchnic tissues was inoculated into the allantoic cavity of 9-day-old duck embryos after filtered through 0.22?m filter.The embryos were killed after three passages and the ELD50 was 10-4.2/0.2m L.The viruses were purified with sucrose density gradient centrifugation and were observed under electron microscopy,a kind of 22 nm virions with classical parvovirus characters was observed by electron microscopy.The signs of SBDS could be reproduced by inoculation of allantoic fluids in 1-day old duckings.This further proved that novel duck parvovirus is an important pathogen of the SBDS.Based on the genome of GPV isolate B,six pairs of primer were designed to amplify the complete genome of NDPV DS15 strain.The sequencing results demonstrated that the full-length genome of NDPV DS15 strain was 5104 nucleotides in length.The full-genome contains two major open reading frames and two inverted terminal repeat(ITR)at the 5' and 3' end of the genome,this is similar to GPV.The DS15 strain genome shared the highest identity(99.8%)with the SDLC01 strain,which is also isolated from SBDS of ducks.The DS15 strain shared 92.6%~97.2% homology with other reference GPVs and 81.2%~85.4% homology with MDPVs.The amino acid sequence of NS1 proteins and VP1 proteins were predicted from the nucleotide sequence of DS15.Compared with classic GPV,DS15 strain and other SBDS isolates showed highest similarity on amino acid sequence and they shared the same amino acid substitutions.The phylogenetic tree displayed that DS15 was clustered into a branch with other SBDS isolates and they were clustered into a distinct subgroup included in the GPV-related virus lineage.In order to develop the polyclonal antibodies to detect NDPV,VP3 gene was amplified by PCR and cloned into the plasmid p GEX-4T-1,then the recombinant plasmid p GEX-VP3 was transformed into E.coli BL21 for expression and induced with 1.0m M of IPTG.NDPV VP3 protein was purified by the purification method of inclusion body and vaccinated into rabbit to produce polyclonal antibodies.The results of Western blot showed that recombinant VP3 protein has good immunogenicity and polyclonal antibodies can be used to detect NDPV.To provide candidate antigens for subunit vaccines of NDPV.VP2 gene was amplified by PCR and inserted into vector p Fast Bac HTA.Then the new plasmid p Fast Bac HTA-VP2 was transformed into DH10 Bac chemically competent cell and target gene inserted into Bacmid plasmid.After white-blue plaque selection,we got the recombinant bacmid Bacmid-HTA-VP2.Then recombinant bacmid transfected sf9 insect cells.The expression of target gene was detected by indirect immunofluorescence and Western blot after 48 hours,the results showed that VP2 protein was successfully expressed in sf9 insect cell.The result of negative staining electron microscope further prove that recombinant VP2 protein of NDPV was able to self-assemble into VLPs with a size and appearance similar to that of wild-type of NDPV virions.Animal experiments showed that VLPs had good immunogenicity for NDPV.This study laid a good foundation of developing subunit vaccine for NDPV.