| Acquired immunodeficiency syndrome(AIDS)caused by human immunodeficiency virus(HIV)infection is one of the most concerned diseases in the world.Currently,its control is mainly through the use of nucleoside reverse transcriptase inhibitors(NRTIs),such as Zidovudine and lamivudine.On the one hand,the use of NRTIs significantly reduces the incidence of AIDS.But on the other hand,the emergence of drug-resistance of HIV under the pressure of drug selection has caused hot concern all over the world.Human’s specificity dictates that we cannot conduct targeted observations in humans to study the mechanism of drug resistance in HIV.At present,a large number of samples were analyzed by sequence analysis before and after treatment,or through some animal models and cell models in vitro.Avian leukosis virus(ALV)was the first virus to be shown to induce tumor.In addition,reverse transcriptase was first discovered in ALV and awarded the 1975 Nobel Prize in Medicine.ALV and HIV are both retroviruses,and they have very similar genomic structure,so we usually use ALV as an excellent model to study the variation of HIV gene and the evolution of quasispecies.Previous studies found that ALV developed resistance to NRTIs during continuous culture and passage of ALV in DF-1 containing NRTIs.Through high-throughput sequencing of a large number of strains,pol gene mutation site related to drug resistance was found,and resistance to ALV was confirmed by infectious cloning and point mutation reversal.So is it easy for ALV to develop drug resistance in SPF chickens infected with ALV in different ways after continuous administration? What are the key mutations in pol gene that determine drug resistance in the body? Are these mutational sites consistent with those found in vitro models? The answers to these questions help answer how ALV evades NRTIs "hunting" in SPF chicken via pol gene mutation and explains its quasispecific evolution law.To avoid the singleness of existing ALV-J infectious clone gene,In this study,six angioma-associated ALV-J wild virus strains were isolated and identified from a Helan brownchicken farm in Shandong province and sequenced the genome.All the 6 strains were7610-7630 nt,and the amino acid homologies of gp85 gene and ALV-J were about 90% while the nucleotide homologies of pol gene were over 97.1%.In order to further study the mechanism of ALV-J drug resistance by gene mutation in vivo,the SDAU1701 strain was inoculated into the yolk sac of chicken embryo at the age of 6 years,The SPF chicken model of persistent viral infection and drug selection in vivo was established by continuous injection of Zidovudine,a retrovirus inhibitor.After hatching,SDAU1701 strains were confirmed to have drug resistance to Zidovudine after long-term monitoring.The high-throughput sequencing technique was used to sequence the two high-mutation regions pol-A(273 bp)and pol-B(266 bp)of the drug target gene pol.The results show that after a long period of Zidovudine selection pressure effect,The pol gene of ALV shows two main mutations,T196A(p < 0.05)and V447A(p < 0.05),which may be related to drug resistance.The in vitro resistance to the mutant clone rSDAU1701(T196A/V447A)was then verified by constructing a mutant of the pol gene mutation.The results showed that the in vitro mutant SDAU1701(T196A/V447A)clearly produced resistance to Zidovudine,and its reverse transcriptase activity was significantly higher than that of the original strain.In this study,the mechanism of drug resistance of ALV-J under the selective stress of Zidovudine in vivo was revealed.The key gene mutations of the virus was found after long-term use of drug action in SPF chicken,which provides a good basis for analyzing the mechanism of drug resistance of retrovirus in animals and establishing a good animal model. |
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