Etiological Investigation Of Avian Leukosis And Analysis On Genetic Polymorphism Of Avian Leukosis Virus Receptor In Huiyang Bearded Chicken

Avian Leukosis?AL?is caused by Avian Leukosis Virus?ALV?or Rous Sarcoma Virus?RSV?of the retrovirus family.In recent years,AL has been spreading in China,and its host range has been expanded from White feather broiler to Meat-type chicken,egg-type chicken,breeding type of chicken and many local chicken breeds in China.The disease in addition to induce benign or malignant tumors,can also result in different sub clinical symptoms,mainly manifested as growth retardation,decreased immune suppression and production performance of poultry industry,causing huge losses.There are no effective vaccines and drugs for the disease at present,mainly through eliminate infected chickens to prevent and control the disease.Huiyang bearded chicken is a famous local chicken variety in Guangdong province.It has been included in the"national animal and poultry genetic resource protection list"revised in 2014,but there has not been a systematic report on avian leukosis infection in Huiyang bearded chicken.Therefore,study on the etiology of huiyang bearded chicken and analysis on genetic polymorphism of avian leukosis virus receptor in Huiyang bearded chicken can provide a scientific basis for the protection of local varieties.In this study,7 representative Huiyang bearded chicken farms in Guangdong province were selected to study on the etiology of avian leukosis.This study collects anticoagulant blood 55,102,46,50,193,105 and 299 samples from 7 farms of A,B,C,D,E,F and G.The samples were inoculated to the DF-1 cells and CEF cells.The positive rates of ALV p27 antigen in these 7 fields based on DF-1 cells were 1.8%?1/55?,21.5%?22/102?,19.6%?9/46?,24%?12/50?,6.7%?13/193?,18%?19/105?,0%?0/299?,and the positive rates of ALV p27 antigen in these 7 fields based on CEF cells were 1.8%?1/55?,27.5%?28/102?,17.4%?8/46?,20%?10/50?,7.7%?15/193?,20%?21/105?,0%?0/299?.In this study,48endogenous viruses and 72 exogenous viruses were isolated from 850 samples.The other six farms all had serious exogenous ALV infection.Meanwhile,endogenous ALV existed in B,C,D,E and F farms.In this study,22 env genes of endogenous ALV and 26 env genes of exogenous ALV were sent to sequenced.Through identification,22 endogenous viruses were ALV-E,and26 exogenous viruses were ALV-J,which was named EGD1701^EGD1722 and JGD1701^JGD1726.The similarity between the isolated strains of E subtype env genes is95.4%¹00%and they are 89.3%⁹9.7%similar to other ALV-E reference strains at home and abroad;The similarity between the isolated strains of J subgroup env genes is89.5%¹00%and they are 91.7%⁹6.8%similar to other ALV-J reference strains at home and abroad.3 base pairs CAA were inserted at the 359 site of the 4 E subtype isolates;3base pairs TCT were inserted at the 627 site of the 7 E subtype isolates;6 isolates of E subtype were transformed from GATAA to TGGGG at the 811 site.Based on the sequence analysis of env gene,we can see that ALV-E or ALV-J from different Huiyang bearded chicken farms have their unique sequence characteristics.In this study,the chicken groups in A and G farms were collected for anticoagulant,31and 37 copies of all blood DNA were extracted from 2 fields.The DNA as a template was used as a template for TVA,TVB,NHE1 sequence amplification,cloning and sequencing.The length of TVA sequence was 1999²009 bp,the length of TVB sequence was 301 bp,and the NHE1 sequence length was 6669⁶676 bp.The genetic polymorphism of the avian leukemic virus receptor gene was analyzed.The results showed that two TVA resistant alleles(TVA^r3,TVA^r4)and one susceptible gene?TVA^s?were found in Huiyang bearded chicken;The frequency of TVA^s,VA^r3 and TVA^r44 in A and G farms is 0.07/0.22,0.41/0.75,0.52/0.03);The Huiyang bearded chicken has a TVB susceptible genotype(TVB^s1);There are 10 mutation sites in NHE1 of Huiyang bearded chicken,respectively,c.11653C>T,c.11917C>T,c.11946C>T,c.13429T>C,c.13438T>C,c.14516A>G,c.14522G>A,c.14654C>T,c.15834G>A,c.17560C>G.However,these sites were all synonymous cSNP,and did not change the amino acid sequence.This study showed that the ALV-J infection of several Huiyang bearded chicken farms in Guangdong province was different.It was necessary to start the purification of avian leukosis in Huiyang bearded chicken,and the presence of ALV-E in some Huiyang bearded chicken farms also meant the selection of correct methods for the detection of avian leukosis.This study found that 2 known TVA alleles in Huiyang bearded chicken population provided scientific basis for disease resistance breeding of Huiyang bearded chicken.