诱发血管瘤型J亚群禽白血病病毒gp85基因的克隆与表达

2007年7月至11月,中国开产前后的商品海兰褐蛋鸡群大面积暴发血管瘤,造成巨大经济损失。将病料接种DF1细胞,通过PCR和间接免疫荧光(IFA)确定此次暴发的血管瘤为J亚群白血病病毒(ALV-J)感染引起。从患病鸡群中分离到5株ALV-J(前4株已经报道),将第5株病毒命名为WS0705。为研究该毒株抗原性的特点,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示WS0705与英国ALV-J原型毒株HPRS-103同源性最高。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacH Tb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的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35kD的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中得到良好的表达,并且其编码产物完全可以被外源性ALV-J的特异性单抗JE9识别,进一步证明本次暴发血管瘤的病原为ALV-J,并为进一步开发ALV-J相关诊断产品奠定了基础。     

禽白血病A亚群病毒gp85的单因子血清制备及其特异性鉴定

【目的】为了研究出一种能够针对A亚群禽白血病的快速特异性诊断试剂。【方法】将A亚群禽白血病病毒(ALV-A)SDAU09E1株接种于DF1细胞上,以感染细胞DNA为模板,通过PCR方法扩增出1023bp的ALV-A-gp85基因。将其正确阅读框架插入表达载体PET-32a(+)中,实现在BL21(Rosetta)宿主菌中表达。将纯化的融合蛋白常规免疫小鼠,制备得抗血清。【结果】实验成功获得52.8kDa的重组融合蛋白,且具有良好的免疫原性。间接免疫荧光试验(IFA)表明该血清可与ALV-A和ALV-B反应,但不与ALV-J反应。【结论】该实验首次在国内外研制出能用于鉴别性检测经典的A/B亚群ALV的单因子血清,可与ALV-J特异性单抗互补作用于外源性ALV感染的鉴别性诊断。我国鸡群同时受经典的ALV-A/B和新出现的ALV-J困扰,鉴别诊断非常必要,研究这种试剂具有较高的实用价值。     

禽白血病病毒J亚群env基因的克隆和序列分析

应用多聚酶链反应（PCR）的方法扩增出ADOL-4817毒株的囊膜蛋白env基因,并克隆进大肠杆菌。经核酸序列分析证明,env基因的大小为1746 bp,其中gp85和gp37由1554 bp组成,可翻译成517个氨基酸,分子量为57.7 D。根据糖基化位点N-X-S/T的特点,发现ADOL-4817的env蛋白有15个潜在的糖基化位点。同源性分析证明,ADOL-4817的env基因与其它ALV-J的env基因序列同源性为88.8%～92.4%,而与外源性ALVs的相应序列的同源性仅为40.5%～51.4%,然而,与内源性的EAV-HP毒株的类env基因的同源性高达91.2%;另外,ADOL-4817毒株的gp37在C末端多了13个氨基酸。这些结果提示,ALV-J的env基因存在广泛的变异性,env基因可能来源于内源性和外源性ALVs的重组。     

Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC<,10200> strain).     

芦花鸡中B亚群禽白血病病毒的分离与鉴定

通过接种DF-1细胞(C/E)系,从山东某地方品系芦花鸡的鸡群中分离到一株外源性白血病病毒(ALV)SDAU09C2。与GenBank中已发表的不同亚群鸡ALV参考株的囊膜蛋白gp85的氨基酸序列比较,表明该分离株与B亚群ALV(ALV-B)2个参考株的gp85的氨基酸同源性最高,均为92.5%;与A、C、D、E亚群ALV的gp85的氨基酸同源性仅在73.2%~87.9%之间;而与J亚群gp85的氨基酸同源性更低至30.3%~32.4%。这是我国地方品系鸡群中第一次分离和鉴定ALV-B及其gp85基因的报道。     

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。     

蛋鸡J亚群白血病病毒的分离鉴定及序列分析

通过接种鸡胚成纤维细胞((CEF)及特异性单抗的间接荧光抗体反应(IFA),从中国商品代蛋鸡群中首次分离到J亚群白血病病毒(ALV-J).对其env基因编码的氨基酸序列及3'-末端(3'-Ter)序列与国内外来源于白羽肉鸡的毒株作了比较分析.结果显示,这两株病毒的gp85基因编码的氨基酸序列与国外5个毒株同源性仅为83.4%～87.3%,与国内来源于白羽肉鸡的8株病毒同源性也仅为86.4%～89.6%.gp37基因编码的氨基酸序列与5个国外毒株同源性为91.8%～97.0%,与8个国内毒株同源性为93.9%～95.9%.另外,国内来源于白羽肉鸡的各毒株的3'-Ter序列在"E"区均有明显缺失,但本次分离的来源于蛋鸡群的毒株在"E"区没有缺失突变.与所列出的13株国内外毒株相比,这两个毒株在3'-Ter的缺失最少,较接近于原型株HPRS-103.显然这两株病毒的来源不同于国内白羽肉鸡.     

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.     

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.     

禽白血病病毒J亚群env基因产物的抗原性分析

用PCR扩增方法将ALV Jenv基因不同片段进行了克隆 ,并构建了env基因片段GST融合蛋白载体。用Westernblot实验证明 ,大肠杆菌表达的不同env基因片段的GST融合蛋白能与相应的单克隆抗体产生特异性反应性 ,单克隆抗体JE9和G2识别的抗原位点位于gp85的氨基酸 6 5～ 1 5 5区域 ,而I45识别的抗原表位位于env基因的另一区域 (1 5 6～ 2 3 3位氨基酸 )。ALV J氨基酸多肽而非糖基化位点决定ALV J的亚群特异性     

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.     

Different quasispecies with great mutations hide in the same subgroup J field strain of avian leukosis virus

Blood samples were collected from a local strain of chickens associated with serious tumor cases in Shandong Province.The samples were inoculated into chicken embryo fibroblast and DF-1 cells for virus isolation and identification,respectively.The inoculated cells were screened for three common chicken tumor viruses.Nine strains of avian leukosis virus subgroup J(ALV-J) were identified,and were designated LY1201‐LY1209.The env gene from the LY1201 strain was amplified and cloned.All nine resultant env clones(clones 01-09) were sequenced,and the gp85 and gp37 amino acid regions were subjected to homology analysis.Clones 01 and 03 had 10 amino acid deletions in the gp85 region compared to the other seven clones,suggesting that at least two quasispecies with obvious mutations coexist in the same field strain.Among these nine clones,three had identical gp85 and gp37 sequences,and were recognized as the dominant LY1201 quasispecies.The amino acid sequence homology of gp37 and gp85 among the nine clones was 98.5%-100.0% and 96.6%-100.0% respectively,suggesting that the gp85 region of the env gene can better display the quasispecies diversity of ALV-J than gp37.     

Genotype analyses of Campylobacter isolated from distinct segments of the reproductive tracts of broiler breeder hens

Campylobacter isolated from feces and from the oviduct of six broiler breeder hens were genotyped by using flaA SVR DNA sequence analyses. A diversity of genotypes was observed among fecal and oviduct isolates. Comparison of isolates from the oviducts of individual hens revealed variable results. In three cases (hen 2, hen 3, and hen 6), analyses indicated that isolates from all regions of the individual hen's reproductive tract were closely related; isolates from hen 1 and hen 4 were diverse. Comparison of the Campylobacter isolates between hens revealed that in two cases, hens 1 and 3 and hens 4 and 6, certain isolates possessed identical flaA SVR sequence types. Comparisons of Campylobacter isolates recovered from a distinct region of the oviduct were found to have increased diversity as sampling progressed down the oviduct. This study further demonstrates that Campylobacter is present within the reproductive tract of breeder hens and that this presence may enable vertical transmission of Campylobacter from the breeder hen to the broiler offspring.     

ALV-J GP37 Molecular Analysis Reveals Novel Virus-Adapted Sites and Three Tyrosine-Based Env Species

Compared to other avian leukosis viruses (ALV), ALV-J primarily induces myeloid leukemia and hemangioma and causes significant economic loss for the poultry industry. The ALV-J Env protein is hypothesized to be related to its unique pathogenesis. However, the molecular determinants of Env for ALV-J pathogenesis are unclear. In this study, we compared and analyzed GP37 of ALV-J Env and the EAV-HP sequence, which has high homology to that of ALV-J Env. Phylogenetic analysis revealed five groups of ALV-J GP37 and two novel ALV-J Envs with endemic GP85 and EAV-HP-like GP37. Furthermore, at least 15 virus-adapted mutations were detected in GP37 compared to the EAV-HP sequence. Further analysis demonstrated that three tyrosine-based motifs (YxxM, ITIM (immune tyrosine-based inhibitory motif) and ITAM-like (immune tyrosine-based active motif like)) associated with immune disease and oncogenesis were found in the cytoplasmic tail of GP37. Based on the potential function and distribution of these motifs in GP37, ALV-J Env was grouped into three species, inhibitory Env, bifunctional Env and active Env. Accordingly, 36.91%, 61.74% and 1.34% of ALV-J Env sequences from GenBank are classified as inhibitory, bifunctional and active Env, respectively. Additionally, the Env of the ALV-J prototype strain, HPRS-103, and 17 of 18 EAV-HP sequences belong to the inhibitory Env. And models for signal transduction of the three ALV-J Env species were predicted. Our findings and models provide novel insights for identifying the roles and molecular mechanism of ALV-J Env in the unique pathogenesis of ALV-J.     

禽白血病病毒J亚群env基因的克隆和序列分析

应用多聚酶链反应（PCR）的方法增出ADOL－4817毒株的囊膜蛋白env基因,并克隆进大肠杆菌。经核酸序列分析证明,env基因的大小为1746bp,其中gp85和gp37mh 1554bp组成,可翻译成517个氨基酸,分子量为57.7kD。根据糖基化位点N－X－S／T的特点,发现ADOL－4817的env蛋白有15个潜在的糖基化位点。同源性分析证明,ADOL－4817的env基因与其它ALV－J的env基因序列同源性为88.8％－92.4%,而与外源性ALVs的相应序列的同源性仅为40.5％－51.4％,然而,与内源性的EAV－HP毒株的类env基因的同源性高达91.2％;另外,ADOL－4817毒株的gp37d C末端多了13个氨基酸,这些结果提示,ALV－J的env基因存在广泛的变异性,env基因可能来源于内源性和外源性ALVs的重组。     

我国1999—2003年间ALV—J野毒株gp85基因变异趋势

通过接种鸡胚成纤维细胞（CEF）、间接免疫荧光试验（IFA）和聚合酶链式反应（PCR）,连续五年从全国各地的送检病料中分离到14株J亚群白血病病毒（ALV-J）。为了动态观察ALV-J囊膜表面结构蛋白（GP85）的变异情况,对这14株野毒株的囊膜糖蛋白基因（env）进行了克隆和测序,将它们与HPRS-103株的GP85的氨基酸序列进行了比较,结果表明：ALV-J的囊膜表面结构蛋白发生了很大的变异,而且这些变异主要集中在高变区hr1、hr2和vr3;这些野毒株GP85的氨基酸序列的同源性在86．6％～100％之间（从同一鸡场中分离到的两株ALV-J即BJ00302与BJ0303的同源性为100％,其它毒株之间的同源性均小于100％）;有义突变与沉默突变的比例显示这3个高变区极有可能是免疫选择压作用的位点。     

Genotype Analyses of Campylobacter Isolated from Distinct Segments of the Reproductive Tracts of Broiler Breeder Hens

Campylobacter isolated from feces and from the oviduct of six broiler breeder hens were genotyped by using flaA SVR DNA sequence analyses. A diversity of genotypes was observed among fecal and oviduct isolates. Comparison of isolates from the oviducts of individual hens revealed variable results. In three cases (hen 2, hen 3, and hen 6), analyses indicated that isolates from all regions of the individual hen's reproductive tract were closely related; isolates from hen 1 and hen 4 were diverse. Comparison of the Campylobacter isolates between hens revealed that in two cases, hens 1 and 3 and hens 4 and 6, certain isolates possessed identical flaA SVR sequence types. Comparisons of Campylobacter isolates recovered from a distinct region of the oviduct were found to have increased diversity as sampling progressed down the oviduct. This study further demonstrates that Campylobacter is present within the reproductive tract of breeder hens and that this presence may enable vertical transmission of Campylobacter from the breeder hen to the broiler offspring. Received: 11 January 2002 / Accepted: 13 March 2002     

Independent Isolates of the Emerging Subgroup J Avian Leukosis Virus Derive from a Common Ancestor

A new subgroup of avian leukosis virus (ALV) that includes a unique env gene, designated J, was identified recently in England. Sequence analysis of prototype English isolate HPRS-103 revealed several other unique genetic characteristics of this strain and provided information that it arose by recombination between exogenous and endogenous virus sequences. In the past several years, ALV J type viruses (ALV-J) have been isolated from broiler breeder flocks in the United States. We were interested in determining the relationship between the U.S. and English isolates of ALV-J. Based on sequence data from two independently derived U.S. field isolates, we conclude that the U.S. and English isolates of ALV-J derive from a common ancestor and are not the result of independent recombination events.     

二株B亚群禽白血病病毒全基因组序列及其在细胞上的复制性比较

本研究比较了从山东地方品系鸡群分离到的二株B亚型禽白血病病毒(ALV)SDAU09E3和SDAU09C2的全基因组序列及它们在细胞培养上的复制动态。这二株ALV-B的同源性为95.4%,与GenBank中3株B亚群参考株之间的同源性也均在91.0%～94.9%间,而与其它亚群参考株的同源性均低于87.9%。与亚群无关的gag、pol基因和LTR的核苷酸序列比较表明,这二株ALV-Bgp85基因的gag和pol基因与所有比较的参考株的同源性均在93%以上。LTR与其他外源性ALV参考株的LTR间的同源性在72.6%～88.3%范围内,但与E亚群内源性ALV的LTR的同源性只有51.5%。然而,这二个ALV-B的LTR的同源性也只有74.8%,远低于其他基因组部分的同源性,特别是它们的LTR的U3区同源性只有68.8%,二者在二个CAAT分布上也显著不同。对这二株ALV-B在DF-1细胞上的复制动态比较表明,它们在细胞培养上清液中的TCID50值非常类似,但SDAU09E3株核衣壳蛋白p27抗原的含量显著高于SDAU09C2株。这表明,同一亚群的不同毒株在复制过程中,所表达的p27抗原量与所形成的具有传染性的病毒量间没有平行关系。这一差异与LTR-U3区的相关性则有待应用感染性克隆技术来做进一步深入研究。