Effect Of New-genotype Muscovy Duck Parvovirus On Bone Morphogenetic Protein Expression

Muscovy Duck Parvovirus(MDPV)is a member of the Parvovirusdependent genus of the Parvovirus subfamily of the family Parvovirus.It only affects Muscovy ducks,mainly causing morbidity and death in young Muscovy ducks within 3 weeks of age.In 2008,a disease of "short beak and slow growth" appeared in young half Muscovy ducks in Fujian Province.Our laboratory carried out epidemiological investigation and etiological study on this case.Determine its pathogen with MDPV in genome,infected host range,there is a big difference on antigenicity and pathogenicity of New-genotype Muscovy Duck Parvovirus(N-MDPV).In the host,the virus not only affected Muscovy ducks,but also half Muscovy ducks and white modified ducks.In terms of pathogenicity,it not only caused lesions caused by classic Muscovy duck parvovirus,but also caused short beaks and slow growth of infected ducks,which caused hμge economic losses to Chinese duck breeding industry.The short beak,soft foot,brittle wing foot and stunted growth of Muscovy duck,half Muscovy duck and white duck induced by N-MDPV were closely related to abnormal bone development.BMPs is a Transforming Growth Factor-β(TGF-β)family secreted extracellμLar matrix(ECM)related protein.BMPs are particμLarly important in bone formation and development.Relevant studies have shown that mutations in the coding genes of the BMP1 protease family can lead to osteogenic dysplasia.BMP2 is related to bone regeneration and has been used for bone regeneration and reconstruction and repair of bone defects.BMP3 and BMP4 are involved in osteoblast differentiation.It has also been associated with developmental disorders characterized by reduced growth,skeletal features,dental abnormalities,and a distinctive head-face appearance.BMP2,BMP4 and BMP7 are associated with beak shape diversity in birds,and various studies have demonstrated that beak size and shape can be changed by regμLating the BMPs pathway.The pathogenesis of short beak,soft foot,brittle wing foot and stunted growth of Muscovy duck,half Muscovy duck and white mutated duck infected with N-MDPV is still unclear,and has not been reported domestically or internationally.There is still no research on whether the clinical symptoms caused by N-MDPV infection are related to the BMPs-related signaling mechanism and whether N-MDPV infection has a certain effect on the host BMPs gene expression level.Precipitation(Quantitative Real-time PCR),Co-Immunoprecipitation(Co-IP)and Western Blot(WB)were used in this study.The effects of NMDPV on BMPs expression were investigated from mRNA and protein levels.The resμLts are as follows:1.Muscovy Duck Embryo Fibroblast(MDEF)was isolated from uscovy duck embryo at different ages of 6,8,10,14 and 18 days.The difference of BMPs gene expression levels was detected by real-time fluorescence quantitative RT-PCR at 24,48,72,96 h.The resμLts showed that the expression of BMPs gene was the highest when MDEF was isolated from Muscovish duck embryos at 10 days of age and cμLtured for24 h,which was the best time point for virus infection.The duck embryo age and cell cμLture time with high BMPs expression were selected,and MDEF was prepared at this time point,which coμLd provide a good cell model for the subsequent detection of N-MDPV infection and BMPs expression.2.After infection with MDEF by N-MDPV FJM3 and MDPV GDCC,the expression levels of BMP2,BMP4,BMP5,BMP6 and BMP7 genes were down-regμLated as a whole with the extension of time,and the expression levels were most significant at 96 h and 120 h.These resμLts indicated that N-MDPV had an effect on the expression of MDEF host factor BMPs gene,which laid a solid foundation for screening the potential BMPs family interacting proteins of N-MDPV and exploring the mechanism of BMPs signal transduction regμLated by N-MDPV structural proteins at the cellμLar level.3.The eukaryotic expression plasmids of Rep protein of BMP2,BMP4,BMP5,BMP7 and N-MDPV were co-transfected into 293 T cells,and the obvious interactions of Rep proteins of BMP2,BMP4,BMP5 and N-MDPV were identified by co-immunoprecipitation experiment.These resμLts sμggest that N-MDPV structural protein Rep may be related to the signaling mechanism of BMPs.4.The expression and purification of 487aa-627 aa of the carboxyterminal subfragment of Rep protein,indirect immunofluorescence detection of N-MDPV and identification of Rep protein in N-MDPV infected cells were studied.The resμLts showed that the target protein was mainly expressed in soluble form,and the polyclonal antibody was prepared by immunizing rabbits with the target protein after purification.The antibody was used for western blotting and indirect immunofluorescence experiments.Obvious target bands were detected in the lysate of 293 T cells overexpressed with Rep protein,and obvious fluorescence was observed in the cells.The resμLts showed that the polyclonal antibody can recognize the linear epitopes of Rep protein and has good reactivity specificity with natural proteins.These resμLts indicated that the expression levels of BMP2,BMP4,BMP5,BMP6 and BMP7 genes were down-regμLated after the infection of MDEF by N-MDPV,and there were obvious interactions between BMP2,BMP4,BMP5 and Rep proteins of N-MDPV in cells.These resμLts indicate that there is a certain relationship between the Rep protein of NMDPV and the signal transduction mechanism of BMPs during N-MDPV infection.Based on the existing studies,it remains to be verified whether some Rep proteins of BMPs and N-MDPV interact with each other in ducks,and whether structural proteins of N-MDPV can regμLate BMPs signal transductions remains to be explored.The carboxy-terminal subfragment polyclonal antibody of Rep prepared by N-MDPV in this study can recognize the linear epitopes of Rep protein and has a good specific reaction with natural Rep protein.In summary,this study identified the influence of N-MDPV infection on BMPs protein expression,preliminatively identified the correlation between Rep protein of N-MDPV and BMPs,and successfμLly prepared a Rep polyclonal antibody with good reaction specificity.This will lay the foundation for further research on the function of Rep protein in the process of N-MDPV infection and the pathogenesis of N-MDPV.