The Function Research Of Mutation Sites In Tvb Receptor Gene Of Subgroup B/D/E Of Avian Leukosis Virus

Avian leukosis is an immunosuppressive neoplastic disease caused by avian leukosis viruses?ALV?,which causes tremendous economic losses in the worldwide poultry industry.As for chicken,the avian leukosis viruses can be divided into six subgroups through A to E and J.Among them,ALV-A,B and J are the most common infection,which has become one of the major diseases that seriously endanger our poultry industry.The traditional eradication work needs to spend a lot of manpower and resources.At present,no effective drugs and vaccine are available,and eradication management strategy and biosecurity can only be used.Selective breeding for chickens genetically resistant to ALV has been recognized as an effective means of controlling avian leukosis.Therefore,it's of great significance to study the prevention and control of avian leukosis from the perspective of genetic resistance.In this study,we have found two natural mutation sites tvbg.3667₃668insAG?c.291₂92insAG?and g.3637₃638ins_A?c.360₃61insA?in receptor gene of subgroup B/D/E of avian leukosis virusin the C hinese local chickens based on the genetic diversity evalutate for tvb gene by our laboratory,namedtvb^r4 and tvb^r5.We also have found that both of the natural mutation sites casused premature termination codon and amino acid mutations.Finally,we conducted a series of experiments to study the function oftvb^r4and tvb^r5.1?We conducted the allelic imbalance test of NMD and detection of tvb gene expression by qPC R,and showed that the tvb^r4and tvb^r5mutations induced the Nonsense-mediated mRN A Decay.And further,we could infer that it also reduced the Tvb protein expression.2?We conducted the expression of Tvb protein in 293-FT cells,and was detected by Western Blot,and showed that the premature termination codon caused by tvb^r4 and tvb^r5mutations cloud produce truncated Tvb receptor protein.3?We conducted the Tvb receptor protein overexpression in the 293-FT cells to test if the Tvb^r4 and Tvb^r5 receptor protein could induce cell death,As expected,the premature termination codon caused by tvb^r4 and tvb^r5 mutations all leading to deletion of the death domain and reduced its killing activity.4?We conducted infection experiments,it was proved that tvb^r4 and tvb^r5 mutations could descreased the infection of Tvb receptor-mediated subgroup B/D/E of avian leukosis virus.5?Finally,We conducted the virus envelope glycoprotein and receptor affinity test,it was proved that the amino acid shift caused by the tvb^r4 and tvb^r5 mutations decreased the affinity of the viral envelope glycoprotein and the receptor,and also revealed the molecular mechanism of reducing the host susceptibility to ALV-B/D/E caused by the tvb^r4 and tvb^r5 mutations.In this study,two natural mutations of tvb receptor gene were found in Chinese local chickens in C hina.Through a series of experiments,it was confirmed that mutant Tvb receptor reduced the susceptibility of host to subgroup B,D and E of avian leukosis virus.And reveals its molecular mechanism of reducing s usceptibility,which provides new ideas and technical support for prevention and control of B/D/E subgroup avian leukosis in C hina,which is of great significance.