Molecular Mechanism Of Inhibition Of Duck Skeletal Development By A Novel Goose Parvovirus Infection

Short beak and dwarfism syndrome(SBDS)is an infectious disease caused by the novel goose parvovirus(N-GPV).Since 2014,outbreaks of SBDS have occurred in major duck breeding areas in Shandong,Henan,Anhui and Jiangsu in China,causing serious economic losses.The typical signs of the disease are short beak,tongue outgrowth and stunted growth,with a typical incidence of 80% to 100% and a mortality rate of 10% to 20%.Bone,as an important tissue organ,plays an important role in calcium and phosphorus storage and immunity.The balance of bone metabolism is a necessary prerequisite for the growth,development and production of ducks and is maintained by a combination of osteoblasts,responsible for bone formation,and osteoclasts,responsible for bone resorption.Previous studies have reported that N-GPV infection severely inhibits skeletal development,but the mechanism by which it inhibits skeletal development is unclear,so this study was conducted to investigate the molecular mechanism by which N-GPV inhibits skeletal development in ducks based on pathogenesis.In this study,we used metabolome sequencing(LC-MS)and transcriptome sequencing(RNA-Seq)to screen for differentially expressed metabolites and genes related to skeletal development,identify key candidate metabolites and genes,and use fluorescent probe detection,indirect immunofluorescence and other molecular biology techniques to investigate the mechanisms associated with N-GPV infection affecting skeletal development in ducks,and to determine the molecular mechanism of N-GPV inhibition of mesenchymal stem cells(BMSC),providing a theoretical basis for the prevention and treatment of duck short-beaked dwarf syndrome.The research is divided into the following four main areas:1.Study on the genetic evolutionary characteristics and pathogenicity of N-GPVIn this study,four N-GPV isolates(AHDS,NMG21,SDLY and SDWF strains)were isolated and identified.Sequence analysis showed that the nucleotide homology between the isolates and the reference strains ranged from 91.8% to 100%.Thirteen and 19 amino acid variant sites were identified in the NS1 and VP1 protein regions of the isolates,respectively,which may have implications for viral nucleic acid replication and pathogenicity.Genetic evolutionary analysis showed that the AHDS isolate was more distantly related to the other three isolates and belonged to a branch with reference strains such as JS1 and QH15.The isolates NGM21,SDLY and SDWF were similar to each other and were in the same branch with reference strains such as HN1 P and SD0402,suggesting that the N-GPV strain has evolved significantly and formed a separate branch of genetic evolution compared to the classical GPV strain.The pathogenicity of N-GPV was investigated by pathology,histopathology and organ viral load testing using the NGM21 strain to infect 1 day old ducklings and goslings respectively.Ducklings infected with the NGM21 strain show mortality,stunted growth and exhibit typical SBDS symptoms.Infected ducks have hemorrhagic intestinal mucosa with necrosis and loss of villi;bleeding in the liver;enlarged gallbladder with internal bile filling.Viral load results show that the virus can rapidly enter the blood circulation and invade various tissues and organs throughout the body,including the liver,spleen,lungs,pancreas and intestines,where it can replicate.In addition,the results of pathogenicity studies in chick geese have shown that N-GPV infection can cause enlargement and haemorrhage in the liver of infected geese;gallbladder enlargement and bile filling;loss of intestinal mucosa and thinning of the intestinal wall.The above results suggest that the evolution and mutation of N-GPV lead to changes in its pathogenicity,and that N-GPV infection mainly leads to impaired growth and development of infected ducks and damage to liver,intestinal tract and other tissues,and that N-GPV is capable of infecting chicks and endangering the safety of waterfowl farming.2.N-GPV infection causes disorders of skeletal metabolism in ducksThe transcription and replication of N-GPV within the host results in the differential expression of multiple metabolites.The results showed that N-GPV infection caused differential expression of nucleotides,phospholipids,amino acids,sugars,lipids and other metabolites in the beaks and tibiae of ducks infected at 21 d.The differential expression of metabolites related to amino acid and nucleotide metabolism was the most significant.The results of the KEGG metabolic pathway analysis showed that the differential metabolites were mainly involved in metabolic pathways,calcium signaling pathways and other metabolism-related pathways.In this study,screening of biochemical indicators related to skeletal metabolism revealed significant differential expression of hormones related to skeletal metabolism such as testosterone and estrone,suggesting that N-GPV virus infection can affect skeletal metabolism by affecting the expression levels of hormones such as testosterone and estrone.The above study,which clarified the differential changes in skeletal metabolite expression after N-GPV infection,suggested the mechanism of action of disordered bone metabolism caused by N-GPV infection,and provided data to support the pathogenic mechanism of SBDS caused by N-GPV.3.Effects of N-GPV on skeletal development and mechanisms of actionThe results of this study showed that N-GPV infection significantly reduced the skeletal index,calcium and phosphorus content and bone density of ducklings,disrupted the balance of bone metabolism and inhibited bone development.In order to investigate the mechanism by which N-GPV infection inhibits skeletal development in ducks,this study used infected duck tibiae as a research object and constructed gene expression profiles of duckling tibiae.1507 and 1825 differentially expressed genes were obtained from 21 d and 35 d tibiae transcripts respectively,mainly involving functions such as cell growth and necrosis,inflammatory response,signal transduction,skeletal immune system and development.In terms of cell injury,differentially expressed genes are mainly involved in apoptosis,cell necrosis and autophagy,and the main signalling pathways involved in the regulation of cell death are PI3K-Akt,ECM-receptor interaction and NF-kappa B.In terms of bone development,differentially expressed genes were mainly enriched in signalling pathways such as transforming growth factor-β signalling pathway,Wnt signalling pathway and PI3K-Akt signalling pathway.After N-GPV infection,host cells mainly recognize the pathogen through Toll-like receptor signaling pathway,NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway.Subsequently,host cells activate cellular immune response and inflammatory response through JAK-STAT,NF-κB,IL-17 and other signaling pathways.Transcriptome results showed that N-GPV infection significantly reduced the relative expression of BMP2,β-catenin,FGF2 and OPG genes in tibiae and increased the expression of RANKL and the ratio of RANKL to OPG,suggesting that N-GPV infection may inhibit osteoblast differentiation through the BMP/Smads signalling pathway,TGF-βsignalling pathway,Wnt/β-catenin signalling pathway and FGF signalling pathway,and enhance osteoclast activity through the RANKL/RANK/OPG signalling pathway,which together inhibit skeletal development in ducklings.The above study clarified the dynamic changes of genes related to cell damage,skeletal development and skeletal immunity after N-GPV infection,and clarified the mechanism of N-GPV infection inhibiting skeletal development in ducks at the transcriptional level,providing data to support the in-depth study of skeletal development and host immune response after N-GPV infection.4.Study on the mechanism of N-GPV inhibition of proliferation and differentiation of bone marrow mesenchymal stem cellsIn order to clarify the mechanism by which N-GPV inhibits skeletal development,bone marrow mesenchymal stem cells(BMSC)were used in this study to conduct an in vitro validation test.It was found that N-GPV infection significantly reduced BMSC cell activity,led to cell necrosis and apoptosis,and inhibited osteogenic differentiation of BMSC cells.In addition,N-GPV infection inhibited the osteogenic differentiation of BMSC cells by affecting the expression of m RNA and protein levels of key genes in signalling pathways such as Wnt/β-catenin,BMP/Smads and FGF.The use of pathway agonists such as Wnt3 a,BMP2 and FGF2 significantly reversed the inhibitory effect of N-GPV infection on osteogenic differentiation of BMSC cells,suggesting that Wnt/β-catenin,BMP/Smads and FGF are key pathways through which N-GPV infection inhibits the osteogenic differentiation of BMSC cells.Meanwhile,the ratio of RANKL and OPG was significantly increased after N-GPV infection of BMSC cells,suggesting that the OPG/ RANKL/RANK signaling pathway is one of the key pathways that inhibit skeletal development after N-GPV infection.More importantly,we screened for key proteins in the N-GPV infection that inhibit signalling pathways such as Wnt/β-catenin,BMP/Smads,and FGF,and showed that NS1 protein can significantly affect the expression of key genes in pathways such as Wnt/β-catenin,BMP/Smads,and FGF,and thus hypothesized that it is a potential inhibitory protein for skeletal development.In conclusion,this study clarified the mechanism of injury in which N-GPV inhibits bone development,discovered and verified the molecular mechanism of N-GPV inhibition of bone development,and revealed the specific mechanism of N-GPV inhibition of osteogenic differentiation of BMSC cells,which provides a theoretical basis for exploring the pathogenic mechanism of N-GPV and will be beneficial to the prevention and treatment of N-GPV infection.