Isolation, Identification And Replication Characteristic Of Novel Goose Parvovirus SD15 Strain

The sick ducks from Shandong province were identified and isolated in the research.And the sick ducks were diagnosed as NGPV infection by PCR identification and gene sequencing.The isolated virus was named NGPV SD15 strain.The phylogenetic tree analysis of NS1 and VP1 gene revealed that the virus was clustered into the clade of goose parvovirus?GPV?.The NGPV SD15 strain were artificially infected on 40 1-day cherry valley ducks,and these ducks were feed for 6 weeks.And the datum of weight and length of beak were collected.At the same time,the tissues of heart,liver,spleen,lungs,kidney,dodecadactylon and rectum were collected to detect the viral copy number.Pathological section and immunohistochemistry of the tissues mentioned above were made to observe the histopathologic change and detect the tissue distribution of NGPV.After three weeks,the ducks became growth retardation and easy to fracture.But protruding tongues wasn't observed all the time.Post six weeks infection,the weight of NGPV SD15 infected ducks was 1.80±0.22 kg,and the length of beak was 5.65±0.35 cm.The weight of control group ducks was 2.21±0.26 kg,and the length of beak was 6.33±0.35 cm.The data showed that the weight and length of beak of NGPV SD15 infected ducks were significantly lower than control group ducks.In addition,no obvious pathological changes were found in internal organs.The viral copy numbers were detected by real-time quantitative PCR?qPCR?,and the results shown that the no obvious difference among these organs.Immunohistochemical?IHC?tests were performed and mouse anti-NGPV virus polyclonal antibody was used in the experiment.And positive signals were detected equably in these tissues.On the other hand,some of liver cells showed mild oedema and vacuolar degeneration.At medulla area of spleen,acini lienalis disappeared and leukomonocyte reduced.NGPV SD15 was inoculated in 9-day duck embryos,and was passaged blindly.After blind-propagation for 3 passages,it was found that SD15 caused 60% duck embryos death at 48-60 hpi.The copy numbers of allantoic fluid were 10^5.0-10^6.5 copies/mL.The NGPV SD15 infected allantoic fluid of fourth passage was used to inoculate 50%density duck embryo fibroblast cells?DEFs?,and passaged blindly.Cytopathic effects?CPE?were observed in DEFs after blind-replication for 4 passages,and viral copy numbers remained at 10^4.5-10^6.0 copies/mL.Meanwhile,the same as DEFs,NGPV SD15 infected allantoic fluid of fourth passage was used to inoculate goose embryo fibroblast cells?GEFs?.While NGPV cannot cause CPE on GEFs.And viral copy numbers went down from 10^5.3 copies/mL to 10^3.8 copies/mL.It made sure that NGPV cannot replicate on GEFs.The cytotoxicity caused by NGPV on DEFs was detected as well,and the data showed that after 96 h NGPV caused obvious cytotoxicity on DEFs.Through indirect immunofluorescence assay?IFA?to research the replication characteristics of tenth passage NGPV on DEFs.Mouse anti-NGPV virus polyclonal antibody was used in the experiment.The results shown that,on DEFs,Viral signals increased significantly from 72 h and reached the peak at 96 h.so,logarithmic phase was at 72-96 h and plateau phase was at96-120 h.Dichroic staining of PE Annexin V and 7-Amino-Actinomycin?7-AAD?was used to determine the apoptosis of DEFs caused by NGPV SD15.The results showed that NGPV caused mainly cells death but didn't induce apoptosis.To sum up,NGPV SD15 was isolated and identified in this study,and was used to conduct artificial infection experiment.The tissue distribution of NGPV and the pathological changes caused by NGPV SD15 was primarily studied.In addition,the replication characteristics of NGPV in vitro had been researched.These studies laid a foundation for future vaccine and molecular biology research on NGPV.