Isolation And Identification Of ALV-K And Development Of Qualitative Standard Samples Of ALV-A/K Antigen And Positive Serum

Avian leukosis virus(ALV)is a kind of retrovirus which can cause a variety of tumors and immunosuppressive diseases in birds.According to the characteristics of the surface Env protein,ALV can be divided eleven subgroups incuding A-K.Subgroup A,B,C,D,E,J and K mainly infect chickens.ALV-E belongs to endogenous ALV,whereas other subroups are exogenous ALV Among them,ALV-K is a novel subgroup of ALV recently isolated and identified in local domestic chickens in China.Notably,the current detection technologies for ALV purification do not work well for the detection of ALV-K due to its weak replication capacity and often res ? Lt in false negative for ALV-K.In addition,compared with different ALV subgroups,the Env protein of ALV-K has some high homology with that of ALV-A,and ALV-K and ALV-A share a common cell receptor.In order to develop ALV-K specific and sensitive diagnostic technology for the efficient purification of ALV,in this study,an isolate of ALV-K virus was first isolated and identified,and the standard antigen and standard antibody for ALV-K and ALV-A were prepared respectively,and their calibrations were carried out1 Isolation and identification of ALV-K and its genome assayIn this study,virus isolation and identification were carried out for chickens suspected with the infection of ALV in a farm from guangdong province.An isolate of ALV-K was isolated and identified by inoc?Lation of DF-1 cells and PCR amplification,and named as GD180913.The whole genome length of GD180913 isolate was 7483bp and its homology with ALV-K reference strain reached to 98.8%.The size of gag,pol and env was 2103bp,2688bp and 1806bp,respectively,and the size of long-terminal repeat sequence was 274bp.Further sequence analysis revealed that the homology of gp85 of GD180913 with that of ALV-A,ALV-B,ALV-C,ALV-D,ALV-E and ALV-J reference strains was only 38.9%-83.9%,while that with ALV-K reference strains reached to 99.1%.The isolation and identification of ALV-K isolate GD18091 3 provided materials and laid a foundation for the further preparation of ALV-K standard antigen and standard antibody.2 Preparation of ALV-A/K antigen qualitative standard samplesIn order to generate the qualitative standard samples of ALV-A and ALV-K antigens,the ALV-A strain AH 10 and ALV-K strain GD180913 were first confirmed by PCR,IFA and ELISA,and then AH 10 and GD180913 were largely amplified in DF-1 cells,and the purity,titer,homogeneity,stability and preservation time of the amplified viruses were calibrated.Our res ?Lts showed that the amplified AH10 and GD180913 did not contaminate with Necastle disease virus NDV,avian influenza virus AIV,chicken egg drop syndrome virus EDS76,goose parvovirus GPV,Marek's disease virus MDV,chicken infectious bronchitis virus IBV,reticular endothelial hyperplasia virus REV,chicken infectious anemia virus CAV,infectious laryngotracheitis virus ILTV,subgroup J avian leukosis virus ALV-J,bacteria and mycoplasma.Moveover,the amplified AH10 and GD180913 with the titer of 104.63 TCID50/100?L and 104.25 TCID50/100?L respectively had good homogeneity and stability.The acquisition and calibration of ALV-A/K antigen qualitative standard samples can provide standard antigens for the diagnosis of ALV-A and ALV-K in the future.3 Preparation of ALV-A/K positive serum qualitative standard samplesIn order to prepare standard antibodies against ALV-A and ALV-K,the identified and puried ALV-A strain AH 10 and ALV-K strain GD180913 were immunized into SPF chickens by intramusc?Lar inoc?Lation,and the chickens were boosten once every 10 days.Blood samples were collected and sera were isolated after 4 and 5 immunization,respectively.The specificity,antibody titer,purity,homogeneity,stability and preservation time of the prepared sera were determined by IFA and ELISA.Our res?Lts showed that the prepared sera against ALV-A and ALV-K had good specificity without cross-react with the NDV,AIV,EDS-76,GPV,REV,MDV,IBV and ALV-J tested.The prepared sera against ALV-A and ALV-K had ELISA titer of 1:2000 and 1:500,respectively,and IFA titer of 1:200 and 1:100,respectively,had no contamination with bacterial and mycoplasma,and had good homogeneity and stability.The acquisition and calibration of qualitative standard samples of positive sera against ALV-A and ALV-K can provide standard antibodies for the diagnosis of ALV-A and ALV-K in the future.