The expression of env proteins that bind to viral cell receptors on avian leukosis virus (ALV)-susceptible cells can block ALV infection. In this study, we constructed a cell line (DF-1/B) by expressing the ALV-B env protein in DF-1 cells. PCR, immune fluorescence assay, Western blot, and immune electron microscopy results showed that the env gene can be stably expressed in DF-1cells and the env protein could be detected on the DF-1 cell membrane. An antiviral experiment concluded that the DF-1/B cell line could be resistant to 1 × 104 TCID50 ALV-B virus infection, but had no inhibitory effect on other subgroup ALV. This means that the DF-1/B cell line is specifically resistant to ALV-B and can be used as a tool for ALV-B diagnosis.

In recent years, cases of avian leukosis virus (ALV) infection have become more frequent in China. We isolated 6 ALV strains from yellow feather broiler breeders in south China from 2014 to 2016. Their full genomes were sequenced, compared, and analyzed with other reference strains of ALV. The complete genomic nucleotide sequences of GD150509, GD160403, GD160607, GDFX0601, and GDFX0602 were 7482 bp in length, whereas GDFX0603 was 7480 bp. They shared 99.7% to 99.8% identity with each other. Homology analysis showed that the gag, pol, long terminal repeats (LTRs), and the transmembrane region (gp37) of the env genes of the 6 viruses were well conserved to endogenous counterpart sequences (>97.8%). However, the gp85 genes displayed high variability with any known chicken ALV strains. Growth kinetics of DF-1 cells infected with the isolated ALV showed viral titers that were lower than those infected with the GD13 (ALV-A), CD08 (ALV-B), and CHN06 (ALV-J) on day 7 post-infection. The infected Specific-pathogen-free (SPF) chickens could produce continuous viremia, atrophy of immune organs, growth retardation and no tumors were observed. These subgroup ALVs are unique and may be common in south China. The results suggested that updating the control and eradication program of exogenous ALV for yellow feather broiler breeders in south China needs to be considered because of the emergence of the new subgroup viruses.

A novel avian leukosis viruses (ALV) subgroup named ALV-K was recently isolated from Chinese indigenous chickens which is different from the subgroups (A to E and J) that have previously been reported to infect chickens. More and more ALV-K strains have recently been isolated from local breeds of Chinese chickens. However, there are no more effective diagnostic methods for ALV-K other than virus isolation followed by envelope gene sequencing and comparison. Viral infection can be blocked through expression of the viral receptor-binding protein. In this study, we have engineered a cell line, DF-1/K, that expresses ALV-K env protein and thereby confers resistance to ALV-K infection. DF-1/K can be used in combination with the ALV-K susceptible cell line DF-1 as a specific diagnostic tool for ALV-K and provides a good tool for further research into the molecular mechanisms of interaction between ALV-K env protein and the host cell receptor.

In this study,we investigated the proliferation of avian leukosis virus subgroup K (ALV-K) and the activity of LTR.An ALV-K strain was used to infect DF-1 cells and the LTR was cloned into pGL3-Basic vector.The replication ability of the virus on ALV susceptible cell DF-1 was evaluated by ALV p27 antigen ELISA,and the promoter activities of the LTR of ALV-K,ALV-E and ALV-J strains were measured using a Dual-Glo Luciferase Assay System.The results showed that promoter activity of ALV-K LTR was significantly lower than those of other exogenous ALV in DF-1,CEF and 293T cells.The relatively poorer replication ability of ALV-K may be related to its weak promoter activity of the LTR.

In order to investigate the role of semen of yellow feather grandparent roosters in avian leukosis (AL) eradication process,a,b,c three different breeds of yellow feather grandparent roosters were chosen to be sampled.ALV p27 antigen ELISA was used in direct detection of cloacal swabs,while anti-coagulated blood and semen sample were used to isolate and detect exogenous ALV based on DF-1 cells.Upon comparative analysis,the results showed that the virus could be isolated from semen,while the result of ALV p27 antigen ELISA S/P value of cloacal swabs and the virus isolation of plasma were negative.Three results couldn't completely correspond respectively.Individual AL deteetion of cloacal swabs,plasma or semen could lead to missed detection.Therefore in AL eradication of yellow feather breeds,semen as the test sample is feasible and should not be neglected,multiple samples should be considered in order to improve the detection rate and speed up the purification process.

To develop an indirect ELISA method for the detection of antibodies against subgroup K avian leukemia virus,the gp85 gene of ALV-K GDFX0603 strain was amplified by PCR,then the prokaryotic expression plasmid pET-GDFX0603-gp85 was constructed.The induction conditions of prokaryotic expression were screened,then the recombinant protein was purified by Ni colum.Using purified recombinant protein as coating antigen,an indirect ELISA method for the detection of antibodies against ALV-K was determined.The results showed that the gp85 recombinant protein was expressed as inclusion bodies,and its size was 53 ku.Western-blot analysis showed that recombinant protein had good immunogenicity.The intra-and inter-assay demonstrated that the coefficient of maximum variation was 10.41％ and 12.57％,respectively.The specific tests showed that there were no cross reactions with the sera against ALV-B,ALV-J,AIV,NDV,IBDV,MDV,REV,Escherichia coli and Salmonella,while it had weak cross reactions with the serum against ALV-A.The results showed that the indirect ELISA method for the detection of antibodies against ALV-K had good reproducibility and better specificity.

为了比较3种ALVp27抗原ELISA试剂盒对外源性ALV检测效果的差异,本研究将1株滴度为4.64×104TCID50/mL的ALV-A毒株GD-13分别以100、10-1、10-2、10-3进行倍比稀释(每个稀释度病毒接种含量分别为2.32 ×103 TCID50、2.32×102TCID50、2.32×101TCID50、2.32TCID50)接种于24孔板的含10％ FBS DF-1细胞悬液中,待细胞长满后换为1％ FBS维持液,从更换维持液后第3天开始至第9天每天取细胞上清分别同时用3种不同的ELISA试剂盒(A、B、C)进行检测.结果显示,当病毒接种含量为2.32×103TCID50时,A、B试剂盒最早可在换液后第3天检出病毒p27抗原阳性,C试剂盒最早在第4天才可以检出病毒阳性,检测阳性率均为100％(5/5).当病毒接种含量为2.32×102TCID50时,使用A试剂盒在接种后第6天能够检出,检测阳性率为80％(4/5);使用B试剂盒最早在接种后第5天可检测出,检测阳性率为20％(1/5);使用C试剂盒直到第9天才能检测出病毒,而且检测阳性率为20％(1/5).当病毒接种含量为2.32×101TCID50和2.32TCID50时,使用A、B和C试剂盒均检测不出病毒.本 研究结果表明,对于ALV-A的检测,无论是在检出时间还是检出率上B试剂盒都优于A、C试剂盒,提示不同商品化试剂盒的灵敏度存在一定的差异.本研究可为禽白血病病原学和净化检测提供参考.

【目的】评估黄羽种鸡禽白血病的净化工作,比较源于同一鸡群不同样品对禽白血病病毒（ALV）检测结果的影响.【方法】采用ALV抗原ELISA方法对广东一黄羽种鸡场采集的2 691枚种蛋进行检测,根据检测结果采集其中70份不同S/P区间所对应的母鸡抗凝血,分离血浆后分别同时接种于CEF和DF-1细胞,用病毒分离方法进行检测,并用PCR方法进行亚群鉴定.【结果和结论】从送检种蛋蛋清共检出ALV p27阳性样品243份,阳性率为9.03%（243/2 691）;蛋清不同ELISA S/P区间所对应的病毒分离情况分别为:蛋清ELISA S/P≥2.0的鸡只其血样CEF和DF-1细胞病毒分离率均为100%;1.5≤S/P＜2.0的鸡只病毒分离率均为88.9%;1.0≤S/P＜1.5的鸡只病毒分离率均为85.7%;0.2≤S/P＜1.0的鸡只病毒分离率分别为60%~75%（CEF）和40%~50%（DF-1）;0.1≤S/P＜0.2的鸡只病毒分离率为62.5%（CEF）和50%（DF-1）;S/P＜0.1的鸡只分别为27.2%（CEF）和9.1%（DF-1）的病毒分离率.PCR结果显示,所有7份CEF+DF-1-的CEF培养物中有6份为内源性ALV-E.研究表明,在蛋清ELISA检测时,S/P越高的阳性蛋清样品其对应母鸡越可能是外源性ALV病毒血症阳性;有一定比例S/P较低的阳性蛋清样品是由对应母鸡体内的内源性ALV排毒所致;而净化初期少数蛋清S/P为阴性的蛋清,其对应母鸡仍可能检出外源性ALV,说明在黄羽种鸡外源性ALV净化方案中单独使用1次蛋清ELISA检测可能会给禽白血病的净化检测造成一定的"误诊"和"漏检".