Studies On The Biological Characteristics And Beak Teratogenic Mechanism Of Novel Goose Parvovirus

The typical clinical symptoms of short beak and dwarfism syndrome are weak feet,short beak and stunting.This disease was first reported in the mule ducks in southwestern France in 1971 and the further outbreak was reported in Poland and Hungary in 1995 and2009,respectively.In 2015,the disease occurred in some parts of Fujian province,then in Shandong,Hebei,Henan,Anhui and Jiangxi,causing huge economic losses in duck farming.Therefore,determining the pathogen of short beak and dwarf syndrome,analyzing the pathogenic characteristics and epidemiology of the disease,and formulating feasible prevention and control measures for the syndrome,are of great significance for the healthy development of duck industry in China.Two NGPVs,named as AH strain and GD strain,were isolated from the ducks with the typical symptoms of SBDS.The two viruses,AH and GD,containing genomes with the length of 5053 nt and 5106 nt,respectively,showed 91.8%-99.9%identity with other NGPVs,89.7%-98.1%identity with the classical goose parvovirus（GPV）,and 79.2%-85.0%similarity with the muscovy duck parvovirus（MDPV）.There was no significant difference between the genome of the AH isolates and the GD isolates.Genome-wide amino acids alignment showed that both AH and GD had 14 consecutive nucleotide deletions.Compared with the classical GPV and MDPV,there were 12 special amino acid mutations in the NS protein of the AH and GD,8 amino acid mutations in the VP protein.Cherry Valley ducks and goslings were infected with AH strain.The typical symptoms of SBDS were observed in Cherry Valley ducks,the beaks of the ducks became shorter so that their tongues were not covered.The SBDS did not occur in goslings group and the control group.The organs of the Cherry Valley ducks and goslings also showed significant differences in the virus titers.6 hours to 28 days post-infection,the virus could be detected in all of the monitored organs from Cherry Valley duck,whereas in goslings the virus was not detected in the pancreas and bursa of fabricius,and with the very lower virus titers in other organs.The results showed that AH strain virus successfully replicated typical SBDS in cherry valley duck,with high titer,but poor adaptability to the original host,goslings.The liver genes expression profile of the Cherry Valley ducks was constructed based on BGIS EQ-500 transcriptome technology platform after the ducks were infected with the AH strain.Analysis of the liver genes expression profile showed that NGPV infection affected the regulation of the calcium metabolism genes.Vitamin D3 hydroxylase-associated protein gene can regulate the function of the vitamin D3 hydroxylase,when its expression was decreased by 2.7 folds during the NGPV infection,the yield of1,25-（OH）₂-D3 also decreased.The calcitonin gene was down-regulated by 5.4 folds,suggesting that NGPV infection might affect the calcium deposition in bone by reducing the expression of the calcitonin gene.Wnt signaling pathway plays central roles in the regulation of the embryonic development and functional differentiation,including bone and cartilage formation.It was also affected by the NGPV infection.Differential gene analysis revealed that NGPV infection resulted in delete espression of R-spondin-2 and Wnt3a gene,2.3 and 20 folds down-regulate expression of LGR5/LGR6,the receptors of Wnt signaling pathway which is activated by R-spondin proteins.DKK1 protein blocked the signal transduction of Wnt signaling pathway by inhibiting the interaction between LRP5/6 and Wnt.It was only found in infected samples,suggesting that the expression of DKK1 protein was related to skeletal dysplasia.Transmembrane protein 198 protein promotes LRP6phosphorylation through casein kinase,which further activates Wnt signal transduction.Differential gene analysis showed that transmembrane protein 198 gene was down-regulated by 30.44 folds in the infected group,which may block the Wnt signal pathway.Retinoic acid is a metabolic intermediate of vitamin A in vivo,and is also a common cancer drug.Cychrome P450 26A1,a key enzyme in the retinoic acid metabolism pathway,was reduced by 24.4 folds in the infected group.To further explore the effect of retinoic acid on the development of SBDS,the retinoic acid concentration in the serum of the ducks was tested.The results showed that the retinoic acid concentration in the serum of NGPV infected ducks was significantly higher than that of control ducks 18 days after infection.Retinoic acid feeding test showed that retinoic acid could significantly inhibit the growth of beak in ducks,and significantly reduced body weight.To sum up,it was speculated that duck-origin NGPV could down-regulate the expression of cytochrome P450 26A1,a key metabolic enzyme of retinoic acid,resulting in the accumulation of retinoic acid in vivo,and thus causing shortening of the beak length and the retardation in the development of ducks.