The Study Of ALV-K Pathogenicity And The Study Of ALV-K Infection Mechanism Mediated By ERK/MAPK Signaling Pathway

Avian leukosis virus（ALV）is a kind of infective retrovirus,which mainly causesbirds organ tumors,immunosuppression,decreased egg production rate and weight loss.In chicken,threre 7 subgroups including A,B,C,D,E,J,and K,among which the K subgroup is an emerging subgroup in recent years.There are two types of ALV-K strains:exogenous LTR strains and endogenous LTR strains.The exogenous LTR strain was only isolated from the samples of the tumor cases in Luhua chicken in China.However endogenous LTR strains have weak replication capacity and there are no reports of tumor cases.Endogenous LTR strains have been reported in different countries.Our transcriptome data in SPF chicken liver samples revealed that ALV-K infection caused the enrichment of the related factors of ERK/MAPK signaling pathway.Therefore,The purpose of this study is to reveal the roles of ERK/MAPK pathway which was actived by endogenous LTR ALV-K in the process of infection.In order to exploze the infection capacity of the endogenous LTR strain GD1601,The ALV-K strain GD1601,isolated from albumen samples of the healthy chicken flocks was studied.The virus titer was significantly lower than the ALV-J CHN06 strain at various monitoring time points in DF-1 cells.In vivo infection test,The cloacal shedding of virus reactionreached the highest positive rate 33.3%（4/12）on 49 d SPF chicken that infected with endogenous LTR ALV-K.There was only one sample was positive on 7 DPI and 14DPI after infected with 10³ TCID₅₀/0.2 mL ALV-K GD1601.Since then,All the chicken viremia were negative via virus isolation.In 49,63,77 and 91 DPI,The RT-PCR test results of total RNA extracting chickens peripheral blood mononuclear cells showed that the positive rate was between 33.3%⁷7.8%,and two were positive among three chickens in cohabitation group.In addition,Compared with other organs,There were a higher virus load in the lungs,kidneys and glandular stomach in vivo infection.It’s worth noting that ALV-K GD1601 strain had no effect on the weight of chickens（P>0.05）,there were no tumor cases during the trial period of 91 days.In order to study the role of ERK/MAPK signal pathways in viral replication,a fluorescent quantitative PCR detection method was developed to quantify the viral RNA.The assay was specific for ALV-K and did not cross-react with other ALV subgroups,avian influenza virus,Newcastle disease virus,or Marek’s disease virus.The method was 100times more sensitive than that of conventional PCR.Moreover,the CV values for both intra-assay and inter-assay tests of ALV-K positive liver tissue samples were less than1.95%,which indicated that the method had a stable repeatability.The relationship between ALV-K and intracellular ERK/MAPK signaling pathway.In vitro,only at the early stage of the virus infection（15 min）,ALV-K caused phosphorylation of ERK2 protein.And the phosphorylation level is dependent on the virus titer,then the phosphorylation level gradually decreases to normal level until 6 h after infection.ALV-K infection also induced the activation of ERK2 protein of chicken spleen cells in vivo.Further study ERK/MAPK signal pathway upstream and downstream proto-oncogene in vitro and in vivo.Preliminary inspection found that 10³ TCID₅₀/0.2 mL ALV-K GD1601up-regulation the upstream proto-oncogene Ras and downstream proto-oncogene c-Myc.It is worth noting that the inhibitor PD98059 and U0126 of ERK/MAPK signal pathway reduce the gene expression of Ras and c-Myc in vitro,and depending on the concentration of the inhibitor.Therefore,we believe that ALV-K can activate ERK/MAPK signal pathway both in vitro and in vivo,and ALV-K can cause proto-oncogenes Ras and c-Myc rise,and the process is dependent on the ERK/MAPK signal pathway activation.We further study the role of ERK/MAPK signaling pathway in ALV-K breeding.Viral RNA synthesis levels,ALV-K p27 protein expression levels were detected using fluorescence quantitative PCR and ELISA after inhibition of ERK/MAPK signal pathway or siRNA interferes with ERK2 protein expression in vitro.The results showed that the virus RNA synthesis and p27 protein expression levels are influenced by inhibitors in intracellular and extracellular levels.We got the same results when ERK2 protein expression was interfered by siRNA interference technology.These indicated that the ERK/MAPK signaling pathway play an important role in the replication of ALV-K.In conclusion,the endogenous LTR ALV-K with low pathogenicity can also activate the ERK/MAPK signaling pathway,and the activation of ERK/MAPK signaling pathway affects the replication of ALV-K in vitro levels.On the other hand,activation of ERK/MAPK signaling pathway up-regulated the upstream proto-oncogene Ras and downstream proto-oncogene c-myc.