NHE1 gene associated with avian leukosis virus subgroup J infection in chicken

As a kind of binding protein, the type 1 Na⁺/H⁺ exchanger (NHE1) is a receptor for the highly pathogenic Avian leukosis viruses-J subgroup (ALV-J) in chicken. In order to investigate the potential effect of chicken NHE1 gene on leukosis, we compared its expression between ALV-J-affected and -unaffected chicken, screened variations across the whole gene, and then performed association analysis with ALV-J affected/unaffected trait in three un-related chicken populations. We found that the NHE1 gene expressed in four immune tissues including spleen, bursa fabricius, liver, and thymus, and its expression was significantly up-regulated in liver and thymus of ALV-J-affected chickens (with leukosis phenotype) compared to -unaffected ones (ALV-J-negative controls). Thirty-six single nucleotide polymorphisms (SNP) were identified in a 6,105 bp region of the chicken NHE1 gene, giving rise to every 170 bp per SNP. Two SNP of g.4405A>G and g.5886C>G were genotyped with PCR-RFLP method. Results showed that g.4405A>G was significantly associated (P < 0.05) with ALV-J infection in all of the three chicken populations, including White Recessive Rock (WRR), Dwarf Yellow (DY) and Shiki Yellow (SY), while g.5886C>G was significantly associated (P < 0.05) with ALV-J infection in SY. These results indicated that the NHE1 gene was related to ALV-J infection in chicken.     

J亚群鸡白血病病毒AH09/2株的gp85基因的克隆与原核表达

利用PCR方法扩增出J亚群禽白血病病毒(ALV-J)AH09/2株的gp85基因全长930 bp DNA片段。经T载体克隆测序并连接到pGEX-6p-1载体上,构建了重组表达质粒pGEX-6P-1-gp85,在IPTG的诱导下进行表达。Western-blot结果分析表明,gp85融合蛋白表达产物分子量大小约61 kDa,并能与ALV-Jenv基因单抗发生特异性反应。这些结果为深入研究GP85蛋白的生物学功能及研制ALV-J检测ELISA试剂盒奠定了基础。     

YXXM 基序对J 亚群禽白血病病毒复制的影响

[目的]毒株NX0101是骨髓瘤病变型J亚群禽白血病病毒,其早期感染细胞能诱导PI3 K/Akt信号转导通路的激活,本文针对NX0101毒株是否存在YXXM基序及其作用进行了探讨.[方法]利用TMpred软件对NX0101毒株囊膜蛋白(Env)的氨基酸序列进行生物信息学分析,通过搭桥PCR方法将YXXM基序相应的核苷酸序列突变后,构建突变质粒并转染DF-1细胞,拯救出YXXM突变体毒株NX0101 mt( Y/F,M/A),利用real-time PCR和ELISA方法检测并比较YXXM突变前后毒株在RNA水平和蛋白水平的复制情况.[结果]NX0101毒株Env胞浆区554 -557位氨基酸存在典型的PI3K结合基序YXXM.YXXM基序突变后,病毒RNA转录水平和病毒蛋白合成水平都显著下降.[结论]YXXM基序对NX0101毒株在体外宿主细胞中复制发挥重要的作用.     

J亚群禽白血病病毒跨膜蛋白gp37基因克隆与表达

禽白血病病毒(ALV)跨膜蛋白(TM)在病毒-细胞融合过程中起关键作用,其蛋白内部存在的许多高度保守序列有可能成为抗病毒的重要靶点。对TM结构和功能的深入研究将为抗禽白血病病毒乃至其它反转录病毒相关制剂的研制提供科学的理论基础。本研究通过PCR从本实验室分离的ALV-J山东汶上毒株获得表达TM的gp37基因,克隆连接pMD18-T载体并测序,从已构建的质粒pMD18-T-gp37中酶切回收gp37基因,构建重组转移载体pFastBacHTb-gp37。利用昆虫杆状病毒表达系统对gp37基因进行表达,通过间接免疫荧光和Western blot检测gp37基因表达产物,间接免疫荧光显示,重组杆状病毒感染的sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的sf9细胞蛋白显示出约21kD的阳性条带。结果表明,gp37基因在sf9细胞内表达成功。     

The follicle promotes the evolution of variants of avian leukosis virus subgroup J in vertical transmission

Avian leukosis virus (ALV) is one of the main causative agent of tumor development, which brings enormous economic losses to the poultry industry worldwide. ALV can be transmitted horizontally and vertically, and the latter often give rise to more adverse pathogenicity. However, the propagation and evolution of ALV underlying vertical transmission remain not-well understood. Herein, an animal model for the evolution of variants of ALV subgroup J (ALV-J) in the vertical transmission was built and different organs from infected hens and plasma from their ALV-positive progenies were collected, and then three segments in the hypervariable regions of ALV (gp85-A, gp85-B, LTR-C) were amplified and sequenced using conventional Sanger sequencing and MiSeq high-throughput sequencing, respectively. The results showed that the genomic diversity of ALV-J occurred in different organs from ALV-J infected hen, and that the dominant variants in different organs of parental hens, especially in follicle, changed significantly compared with original inoculum strain. Notably, the dominant variants in progenies exhibited higher homologies with variants in parental hens’ follicle (88.9%–98.9%) than other organs (85.6%–91.1%), and most consistent mutations in the variants were observed between the progenies and parental hen’s follicle. Furthermore, HyPhy analysis indicated that the global selection pressure value (ω) in the follicle is significantly higher than those in other organs. In summary, an animal model for vertical transmission was built and our findings revealed the evolution of variants of ALV in the process of vertical transmission, moreover, the variants were most likely to be taken to the next generation via follicle, which may be related to the higher selection pressure follicle underwent.     

Acutely transforming avian leukosis virus subgroup J strain 966: defective genome encodes a 72-kilodalton Gag-Myc fusion protein

Avian leukosis virus subgroup J (ALV-J), the most recent member of the avian retroviruses, is predominantly associated with myeloid leukosis in meat-type chickens. We have previously demonstrated that the acutely transforming virus strain 966, isolated from an ALV-J-induced tumor, transformed peripheral blood monocyte and bone marrow cells in vitro and induced rapid-onset tumors, suggesting transduction of oncogenes (L. N. Payne, A. M. Gillespie, and K. Howes, Avian Dis. 37:438-450, 1993). In order to understand the molecular basis for the rapid transformation and tumor induction, we have determined the complete genomic structure of the provirus of the 966 strain. The sequence of the 966 provirus clone revealed that its genome is closely related to that of HPRS-103 but is defective, with the entire pol and parts of the gag and env genes replaced by a 1,491-bp sequence representing exons 2 and 3 of the c-myc gene. LSTC-IAH30, a stable cell line derived from turkey monocyte cultures transformed by the 966 strain of ALV-J, expressed a 72-kDa Gag-Myc fusion protein. The identification of the myc gene in 966 virus as well as in several other ALV-J-induced tumors suggested that the induction of myeloid tumors by this new subgroup of ALV occurs through mechanisms involving the activation of the c-myc oncogene.     

J亚群禽白血病病毒NX0101株的TCID50与p27抗原之间的相关性研究

为了研究J亚群禽白血病病毒在细胞上接种后的半数细胞培养物感染量(TCID₅₀)与p27抗原的酶联免疫吸附试验（ELISA）的S/P值之间的相关性,并探讨其意义。本试验将J亚群禽白血病病毒NX0101株接种鸡胚成纤维细胞（CEF细胞）,换维持液后连续10d取样,检测10d的TCID₅₀值与p27抗原的S/P值之间的相关性。同时,将该毒株在DF-1细胞系上传代至20代,取其中的第1代、第5代、第10代、第15代和第20代分别进行TCID₅₀滴度的测定和p27抗原检测。结果表明:在CEF细胞上接种的NX0101株J亚群禽白血病病毒连续10d的TCID₅₀值与p27抗原之间存在显著的相关性（r=0.85277;P<0.0001）呈正相关;在DF-1细胞系上传的不同代数之间也呈显著正相关（r=0.93000;P=0.0220）。由此可以推测J亚群禽白血病病毒的TCID₅₀与p27抗原呈显著正相关,因而可以用ELISA法测得的p27抗原的S/P值对病毒的TCID₅₀值进行估测。     

不同病变型J亚群禽白血病病毒LTR启动子序列分析及活性比较

从ALV-J中国地方分离株SCAU-HN06株(血管瘤病变型)、NX0101株和JS-nt株(骨髓瘤病变型)病毒的细胞培养物提取前病毒DNA,通过PCR扩增各毒株的LTR并克隆,随后进行测序分析。与国内外ALV-J参考毒株LTR序列比较发现:国内地方分离株与英国ALV-J原型株HPRS-103和美国ALV-J原型株ADOL-7501的LTR核苷酸序列相似性为88.0%~97.2%;LTR中的U5区及R区具有较高的保守性,而U3区内存在较大差异。将不同病变型ALV-J的LTR片段分别插入pCAT-basic载体CAT报告基因5'端。用所得的重组报告基因表达质粒转染DF-1细胞,48h后通过测定转染细胞中的CAT表达量来评价LTR启动子的活性。结果表明,SCAU-HN06株与骨髓瘤病变型ALV-J(JS-nt株,NX0101株)LTR启动子活性差异不显著。     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%–99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%–96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110–120, aa#141–151 and aa#189–194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%-99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%-96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110-120, aa#141-151 and aa#189-194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

J亚群白血病病毒NX0101株感染性克隆化病毒的构建及其致病性

采用PCR方法分3段扩增出J亚群白血病病毒NX0 10 1株的前病毒cDNA ,PCR产物经克隆后顺次连接,获得一个含有完整ALV J前病毒cDNA的重组质粒,命名为pALV J NX。将此质粒DNA纯化后转染鸡胚成纤维细胞,以针对ALV J的单克隆抗体JE9对转染后的细胞作间接免疫荧光反应,证明获得了具有感染性的病毒。测定原始野毒和分子克隆化病毒的半数组织感染量(TCID50 ) ,分别人工接种1日龄商品代肉鸡并隔离饲养17周。接种野毒组死亡率为2 6 % ,髓细胞瘤发病率为2 4 %。接种分子克隆化病毒组死亡率为2 2 % ,髓细胞瘤发病率为2 2 %。结果表明,克隆化病毒具有天然病毒的致病性并对肉用型鸡表现致瘤性     

Differentially expressed genes in a flock of Chinese local-breed chickens infected with a subgroup J avian leukosis virus using suppression subtractive hybridization

Avian leukosis virus subgroup J (ALV-J) is a new type of virus that mainly induces myeloid leukosis (ML) in chickens. To further elucidate the pathogenesis of ALV-J infection and tumor development, expression profiles from the bone marrow tissue of 15 infected and 18 non-infected birds from a local-breed poultry-farm under naturally infected conditions, were analyzed by suppression-subtractive hybridization. The birds were diagnosed as ML+ (or ML-) by specific ALV-J detection methods, involving serological tests for antigens and antibodies, and RT-PCR to detect viral RNA. A total of 59 partial gene sequences were revealed by differential screening of 496 forward and 384 reverse subtracted cDNA clones. Of these, 22 identified genes, including 8 up-regulated and 14 down-regulated, were related to immune functions, these genes being, MHC B-G antigen, translationally-controlled tumor protein (TPT1/TPTC), transferrin and ferritin, hemoglobin and Carbonic anhydrase. Four of the down-regulated genes were selected for further analysis, in view of their predicted roles in infection and immunity by real-time qRT-PCR, using RNA collected from the same birds as those used for SSH. The four genes were expressed at significantly lower levels (p < 0.001) in ALV-J infected birds than in non-infected ones.     

致蛋鸡血管瘤J亚群禽白血病病毒cDNA全序列分析

【目的】了解近年来我国商品蛋鸡群中以血管瘤为主要表型的J亚群禽白血病病毒(Avian Leukosis Virus subgroup J,ALV-J)的分子生物学特性,为控制ALV-J在鸡群中流行提供基础资料。【方法】采用PCR扩增和序列分析技术,对分离自血管瘤或者血管瘤与髓样细胞瘤(Myeloid Leukosis,ML)并存的3株蛋鸡ALV-J毒株前病毒DNA的全序列及3株商品蛋鸡血管瘤型分离毒和1株商品蛋鸡ML型分离毒的致瘤关键性序列进行研究。【结果】来自血管瘤或者血管瘤与ML并存的商品蛋鸡分离毒株与来自肉鸡分离毒株的全序列差异明显,在遗传进化树上分属两个大的分支;研究发现商品蛋鸡血管瘤及ML混合病例分离毒JS09GY3与JS09GY6株的引物结合位点(Primer Binding Site,PBS)-Leader中出现极为罕见的连续19bp的插入突变,其与劳斯相关病毒1(Rous Associated Virustype1,RAV-1)、劳斯相关病毒2(Rous Associated Virustype2,RAV-2)及劳斯肉瘤病毒施密特-鲁宾二氏[Rous sarcoma virus(strain Schmidt-RuppinB),RSV-SRB]毒株序列相同;通过对U3区调控元件的分析,发现血管瘤商品蛋鸡病例分离毒NHH与JS09GY5的U3区各发生1处连续序列缺失,出现了极为独特的c-Est-1、TCF11及C/EBP结合位点,这些调控元件可能与病毒的致肿瘤特性相关;所测的5株血管瘤商品蛋鸡分离毒均保留完整E元件,而所有肉鸡分离毒的E元件均发生了几乎相同的大部分序列缺失;首次发现血管瘤商品蛋鸡分离毒JS09GY3的E元件中有11bp的连续插入序列。【结论】商品蛋鸡血管瘤型ALV-J与肉鸡分离毒在全序列上差异明显,U3、DR1和E元件等区域有一部分特殊的突变与毒株的宿主类型和肿瘤表型有一定关系,其功能尚需进一步研究。而血管瘤型、髓细胞瘤型ALV-J可能是ALV-J与其它反转录病毒的重组毒。     

Mutational analysis of the subgroup A avian sarcoma and leukosis virus putative fusion peptide domain

Short hydrophobic regions referred to as fusion peptide domains (FPDs) at or near the amino terminus of the membrane-anchoring subunit of viral glycoproteins are believed to insert into the host membrane during the initial stage of enveloped viral entry. Avian sarcoma and leukosis viruses (ASLV) are unusual among retroviruses in that the region in the envelope glycoprotein (EnvA) proposed to be the FPD is internal and contains a centrally located proline residue. To begin analyzing the function of this region of EnvA, 20 substitution mutations were introduced into the putative FPD. The mutant envelope glycoproteins were evaluated for effects on virion incorporation, receptor binding, and infection. Interestingly, most of the single-substitution mutations had little effect on any of these processes. In contrast, a bulky hydrophobic substitution for the central proline reduced viral titers 15-fold without affecting virion incorporation or receptor binding, whereas substitution of glycine for the proline had only a nominal effect on EnvA function. Similar to other viral FPDs, the putative ASLV FPD has been modeled as an amphipathic helix where most of the bulky hydrophobic residues form a patch on one face of the helix. A series of alanine insertion mutations designed to interrupt the hydrophobic patch on the helix had differential effects on infectivity, and the results of that analysis together with the results observed with the substitution mutations suggest no correlation between maintenance of the hydrophobic patch and glycoprotein function.     

Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.     

中国麻鸡中发现禽J亚群白血病

首次报道了中国特有鸡种——麻鸡J亚群白血病的发病情况。山东某种鸡场饲养的中国麻鸡,于开产前出现消瘦、贫血、瘫痪等症状,死亡率达10%。经大体剖检发现,病鸡的内脏器官均弥漫性肿大,色彩斑驳,质度较硬;在胸骨内侧、小肠浆膜面和气管粘膜面出现大小不等的肿瘤结节,呈灰白色。组织学检查发现,增生的肿瘤细胞为均一的髓细胞。用禽白血病病毒J亚群（ALV_J）的特异性引物进行PCR检测,阳性率为89%(15/17);PCR产物测序,其基因序列、预期氨基酸序列与ALV_J原型株HPRS_103的同源性分别为98.05%和97.4%。用ALV_J单克隆抗体,经免疫组织化学检测发现,在肿瘤组织、肝、脾、肾、骨髓、腺胃中呈现强特异性染色。上述检测表明此髓细胞肿瘤是由ALV_J感染引起的。ALV_J麻鸡病例的发现警示：应注意中国地方种鸡的白血病净化工作。     

Avian leukosis virus subgroup J infection influences the gut microbiota composition in Huiyang bearded chickens

Avian leukosis virus (ALV) poses a major threat to poultry. The chicken gut microbiota plays critical roles in host performance, health and immunity. However, the effect of viral infection on the microbiota of Chinese local chickens is not well understood. In this study, we performed high-throughput 16S rRNA gene sequencing and evaluated the gut microbiota profiles using faeces from ALV subgroup J (ALV-J)-infected and healthy Huiyang bearded chickens (Chinese local chickens). At the phylum level, ALV-J infection mainly increased the abundance of Bacteroidetes and Proteobacteria and decreased that of Firmicutes. An analysis at the order, family and genus levels showed that the abundance of Lactobacillales, Lactobacillaceae and Lactobacillus was the highest in normal chicken faeces, accounting for 89·07%, 86·47% and 86·46%, respectively, of phylotypes. Moreover, samples from ALV-J-infected chickens were enriched with Bacteroidales, Clostridiales, Bacteroidaceae, Ruminococcaceae, Lachnospiraceae and Bacteroides. Our findings highlight that ALV-J infection alters the gut microbiota and disrupts the host–microbial homeostasis in chickens, which may be involved in the pathogenesis of ALV-J infection.