Isolation,Identification And Distribution Of Avian Encephalomyelitis Virus In Experimentally Infected Quails

Avian encephalomyelitis(AE)caused by avian encephalomyelitis virus(AEV)is a contact epidemic,which mainly infracts the nervous system of young birds.AEV is a non-coated single stranded stranded RNA virus,belonging to the Picornavirus family within the genus Tremovirus.At present,the disease can only rely on vaccine immunization to prevention and control,therefore,the use of sensitive and accurate method to determine the dynamic distribution of AEV in quails is very important for AE pathogenicity research and prevention and treatment.In this study,the AEV Xianyang strain was isolated and identified,and the dynamic distribution of AEV XY/Q-1410 in orallyl-infected 1-day-old quails was determined by fluorescence quantitative PCR and immunohistochemistry.The results were as followings:Quails of AE suspected from Xianyang were collected in October 2014,the pathogens were isolated and identified by chicken embryo passage,RT-PCR amplification,neutralization test and animal regression test.RT-PCR results showed that the 288 bp fragment amplified by AEV-specific primers shared 84.9% ~ 99.3% nucleotides similarity and 95.8% ~ 98.9% derived amino acid similarity with AEV reference strains;genetic evolution analysis showed that the strain was located in the same branch with SX strain;neutralization test results showed that the neutralization index was 102.32,which was greater than 50 and proved to be positive;animal regression test results show that the group of infected quails and chicks appeared typical clinical symptoms of AE.The morbidity and the mortality in infected quails were 55.1% and 100%,respectively.The morbidity and the mortality in infected chicks were 60% and 58.3%,respectively.Pathological examination revealed that brain tissues of sick birds softening,cerebral hemisphere contour blurred,no clinical symptoms and pathological changes were observed in the control group.In this study,we isolated a strain of avian encephalomyelitis virus from quail,named AEV XY/Q-1410.Establishment of standard curve.The standard curve was obtained by the linear regression analysis of the logarithmic concentration and number of cycles which was generated by the TILONG 988 fluorescence quantification system for 10-fold ratio of recombinant plasmid(102 ~ 108 copies/?L).The slope of the curve is-3.325 and the correlation coefficient(R2)is 0.9978.The detection limitation was 100 copies.The viral load in different tissues was detected by qPCR assay.qPCR was used to detect viral load in cerebrum,cerebellum,proventriculu,intestine,liver,pancreas,spleen,bursa,lung and kidney of quails infected by AEV XY/Q-1410 at 3–17(one-day intervals),18–25(every day),29,30,32,36–48(one-day intervals),49 and 51–60(every day)days post-infection(dpi),respectively.qPCR results show that the virus made persistent infection up to 60 days in most tissues.The viral load in cerebrum and cerebellum gradually increased to the first peak at 18 dpi and the second peak appeared at 51 dpi and 46 dpi,respectively.The viral load in proventriculus,intestine,spleen and bursa was higher than that in cerebrum and cerebellum.The viral load in proventriculus was "U" shape from 3 dpi to 46 dpi and gradually decreased to 52 dpi,and then remained stablely until 60 dpi.The viral load in intestine was "M" shape from 3 dpi to 46 dpi and remained stableiy until 60 dpi.The viral load in liver fluctuated strongly,and the peak appeared at 52 dpi.The viral load in pancreas was alwalys lower than 100 copies.The viral load in spleen was "W" shape from 3 dpi to 54 dpi and decreased slowly until 60 dpi.The viral load in bursa was "U" shape from 3 dpi to 44 dpi and decreased until 49 dpi,then fluctuated slightly until 60 dpi.The viral load in lung was two continuously "M" shape.The viral load in kidney was only one peak appeared at 7 dpi,and remained slightly fluctuated from day 15 dpi to 60 dpi.Immunohistochemistry was used to detect tissue distribution of AEV.The immunohistochemistry was used to detect the distribution of virus particles in the organs collected at 7 dpi,18 dpi,36 dpi,54 dpi and 59 dpi,meanwhile,Image-Pro Plus software was used to calculate the relative expression of viral protein.The results showed that all tissues had significant yellow staining,whereas negative controls did not.The correlation analysis between the relative expression of viral protein and viral load in organ showed that they are positively correlated.In conclusion,the AEV XY / Q-1410 strain was most closely related to the SX strain,and it was far from the relationship to L2 Z strain,YL strain,1143 strain,Van Reokel strain and Pf-CHK1 / AEV strain,indicating some differences in genetic evolution.The results of qPCR showed that virus made persistent infection up to 60 days and the viral load in proventriculus,intestine,spleen and bursa of was higher than that in cerebrum and cerebellum.The results of correlation analysis showed that the relative expression of viral protein in each tissue was positively correlated with the copy number of viral gene.