Subtype Identification And Molecular Characteristics Of Avian Leukosis Virus Prevalent Strains Of Local Chicken Breeds In Guizhou Province

Avian leukemia vertical(ALV)is a kind of transmission disease caused by avian leukemia virus,can cause the chicken group to produce the benign and the malignant tumor,causes its production performance to drop and the immunity suppression.ALVs are divided into ten subgroups(A-J)based on the virus and host range associated antigenicity of envelope proteins and subgroup K which was identified recently.Avian leukosis subgroup J is a kind of tumorigenic infectious disease with myelocytoma which is caused by avian leukosis virus subgroup J(ALV-J),Its occurrence rate is highest,cause huge loss to the chicken industry.So far,there is no drug that can be prevented and treated,to reduce the virus on the chicken industry hazards,continuous strengthening of the provincial and municipal local chicken group AL purification work has become the most important.In recent years,ALV is not only popular in Roman hens,laying hens,and widely spread in different chicken breeds in every area of our country,affecting the healthy development of local chicken breeds in China.Guizhou Province in the eastern part of the Yunnan-Guizhou Plateau,Water and grass abundant,ecological climatic conditions are superior,gave birth to the Qiandongnan small chicken,Weining chicken,Zhuxiang chicken and Lvkedan chicken and other fine Guizhou local breed chicken,is a valuable resource for the chicken resource gene bank in China.The study found,China in 2005 and 2008 on some local large-scale chicken farms in Guizhou Province,AL-related reports,however,in recent years,the etiology of AL in local breed flocks in Guizhou province has been less studied.In order to understand the AL infection and the main epidemic subtypes of local breeds in Guizhou province in 2016,this study was to collect chicken eggs samples from 9 local breeds in eight regions of Guizhou from February to April in 2016,the etiological detection of ALV and the main epidemic subtypes were carried out,carries on the ALV the pathogen detection and to determine its main epidemic subtype,completes to the Guizhou province local breed chicken ALV infection situation inquiry.In order to understand the ALV infection in Guizhou province,Using ALV-P27 antigen ELISA detection kit 380 eggs of 9 local breeds in eight areas of Guizhou were tested.Collection egg white,the ALV-P27 antigen detection kit was used detection of egg white ALV group specific P27 antigen,The results show Guizhou Province in eight areas of egg white ALV-P27 positive rate antigen was 14.7%(56/380).Among them,Anshun ? Zunyi ?Liupanshui?Changshun?Qiandongnan five regions of the breed have avian leukemia infection,Guiyang?Qianxinan?Bijie three regions of the breed not found avian leukemia infection,however,from Anshun?Changshun?Zunyi three regions of the lvkedan ALV positive rate high,for 26.7%~36%.The ALV-P27 antigen ELISA detection kit is positive for 56 samples,after inoculation of CEF and DF-1 cells respectively,using ELISA?IFA?PCR and egg white extraction of RNA RT-PCR method was used for repeated validation,To explore the results of different experimental methods of the same batch of ALV positive samples.The results show,DF-1 and CEF cells tested the same,fifteen samples were positive,the positive rate was 26.8%(15/56),indicating the presence of exogenous ALV,the detection rate of ALV-P27 antigen in cell supernatant was decreased,probably because in the cell culture process,the amount of virus in the supernatant of cell culture could not reach the minimum virus amount of ALV-P27 antigen detected by ELISA method.these results provide important data for the further improvement of avian leukemia purification methods,we suggest that when AL is purified,if the conditions permit should increase the PCR method of detection,will help reduce the missed rate of ALV.At the same time,Both Trizol and Tissue RNA Extraction Kit were used to extract egg white RNA,will extract a good RNA reverse transcription into cDNA,PCR was performed not detected ALV,may be due to egg white RNA virus content in less,this method is not currently available for the production of positive samples of the review.The egg white ELISA kit to detect ALV-P27 antigen positive samples,respectively,inoculated with CEF and DF-1 cell culture to isolate the virus,after 7 days,the ALV-P27 antigen in the supernatant was detected?infected cells with cDNA Four pairs of ALV primers were used for PCR identification and typing.Cloning and sequence analysis of gp85 gene were carried out in partial ALV isolates of 9 flocks.The results show,The positive rate of cell culture supernatant of 56 positive cells was 26.8%(15/56);fifty-six positive inoculations of CEF and DF-1,all of which amplified ALV,and identified subtypes of all subtypes J(ALV-J).The sequencing of 21 ALV-J gp85 gene homology comparison shows that,98.4% to 100%between isolates,and 86.6% ~ 100% between ALV-J reference strains at home and abroad;phylogenetic tree analysis showed that,21 strains of ALV-J isolates in different branches,the relationship between evolution and the source of no obvious law.This study supplemented the epidemiological survey data of local breeder chickens,which is of great reference significance to the purification and prevention of AL in Guizhou Province.In this study,different methods were used to detect the same ALV positive samples,which provided a powerful basis for speeding up the purification process of avian leukemia in poultry farm and SPF of vaccine products.