Expression of endogenous retrovirus ev/J <Emphasis Type="Italic">gp85</Emphasis> 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 leukosis 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 IMC₁₀₂₀₀ strain). Foundation items: Natural Science Foundation of China (30460098); China Postdoctoral Science Foundation funded project (2005038585).     

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。     

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     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

禽白血病病毒J亚群囊膜蛋白env基因的克隆和表达

禽白血病病毒J亚群（ALV－J）是90年代鉴定出的ALV的新亚群,其囊膜蛋白env基因序列别与ALV A－E亚群的有相当大的差别。为ALV－J env基因及春表达产物的特点,用PCR方法扩增出ADOL－4817毒株的env基因,并克隆进TA载体,经电泳鉴定大小为1.7kb。将克隆出的env基因与杆状病毒pBlue-Bac4表达质粒DNA连接,构建成转移性载体pBac4817env,通过与Bac-N-Blue杆状病毒DNA共转染,区得了重组病毒rBac4817env-2。该重组杆状病毒感染Sf9细胞,能高效表达env基因产物,免疫荧光分析结果证明,单克隆抗体G2或多价兔抗env gp37血清能识别Sf9细胞,能高效表达env基因表达的特异性抗原;Western blotting分析结果表明,表达的重组基因产物的分子量大小约为90kD-94kD。用这些重组基因产物免疫鸡可以诱导鸡导鸡产生出高滴度的抗ALV－J特异性抗体。这一结果提示,这种杆状病毒表达的重组基因产物有助于ALV－J env基因生物学特性的深入研究。     

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。     

Segregation of EAV-HP Ancient Endogenous Retroviruses within the Chicken Population

Avian leukosis virus subgroup J (ALV-J), an exogenous avian retrovirus, is thought to have evolved by recombination with the highly identical env gene of the endogenous avian retrovirus EAV-HP. Embryonic expression of EAV-HP env has been suggested to be associated with the induction of immunological tolerance, a feature observed in a significant proportion of meat-type chickens infected with ALV-J. In support of this hypothesis, we demonstrate that EAV-HP loci, some of which could be associated with tolerance, are still segregating within the chicken population.     

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。     

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.     

Genome structure and expression of the ev/J family of avian endogenous viruses.

We recently reported the identification of sequences in the chicken genome that show over 95% identity to the novel envelope gene of the subgroup J avian leukosis virus (S. J. Benson, B. L. Ruis, A. M. Fadly, and K. F. Conklin, J. Virol. 72:10157-10164, 1998). Based on the fact that the endogenous subgroup J-related env genes were associated with long terminal repeats (LTRs), we concluded that these LTR-env sequences defined a new family of avian endogenous viruses that we designated the ev/J family. In this report, we have further characterized the content and expression of the ev/J proviruses. The data obtained indicate that there are between 6 and 11 copies of ev/J proviruses in all chicken cells examined and that these proviruses fall into six classes. Of the 18 proviruses examined, all share a high degree of sequence identity and all contain an internal deletion that removes all of the pol gene and various amounts of gag and env gene sequences. Sequencing of the gag genes, LTRs, and untranslated regions of several ev/J proviruses revealed a high level of identity between isolates, indicating that they have not undergone significant sequence variation since their introduction into the avian germ line. Although the ev/J gag gene showed a relatively weak relationship (46% identity and 61% similarity at the amino acid level) to that of the avian leukosis-sarcoma virus family, it retains several sequences of demonstrated importance for virus assembly, budding, and/or infectivity. Finally, evidence was obtained that at least some members of the ev/J family are expressed and, if translated, could encode Gag- and Env-related polypeptides.     

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。     

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     

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。     

鸡的J亚群白血病病毒的分离及部分序列比较

通过接种鸡胚成纤维细胞、聚合酶链式反应（ＰＣＲ）技术及特异性单抗的间接荧光抗体反应（ＩＦＡ）,从某大型肉用型种鸡场的疑似Ｊ亚群白血病的病鸡中,以及２５个临床健康的商品代肉鸡群的２群中,分离鉴定出Ｊ亚群禽白血病病毒（ＡＬＶ－Ｊ）。在用抗ＡＬＶ－Ｊ ｇｐ８５单克隆抗体ＪＥ９的ＩＦＡ中,来自病鸡群的两株病毒ＳＤ９９０１和ＳＤ９９０２呈强阳性反应,来自临床健康肉鸡群的ＹＺ９９０１和ＹＺ９９０２株呈弱阳性反     

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.     

Analysis of precursors to the envelope glycoproteins of avian RNA tumor viruses in chicken and quail cells.

Immune precipitation with monospecific antiserum was employed to study the intracellular synthesis of viral glycoproteins gp85 and gp37. Labeled gp85 and gp37 were detected from lysates of cells transformed with Rous sacroma virus, strain B77, after long-term labeling with radioactive glucosamine or phenylalanine. Immune precipitates prepared from lysates of cells pulse-labeled for a short time resulted in a glycoprotein of 92,000 molecular weight (gp92). This precursor was stable in B77-transformed Japanese quail cells for several hours, whereas in chicken cells it could be chased within a few hours into virion glycoproteins gp85 and gp37. Similarly, the precursor for the structural viral proteins, pr76, persisted in quail cells much longer than in chicken cells. During very short pulses or in the presence of a glucosamine block (25 mM glucosamine), the antiserum against the viral envelope glycoproteins detected a precursor of higher electrophoretic mobility of approximately 70,000 molecular weight, "p70." Fucose label entered gp92 and gp85 as well as "p70." Proteolytic treatment of virion-bound gp85 in vitro generated two discrete glycoproteins of 62,000 and 45,000 molecular weight, but did not result in an increase in the amount of gp37.     

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。     

env Gene of Chicken RNA Tumor Viruses: Extent of Conservation in Cellular and Viral Genomes

The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNA(gp)) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA(gp) and chicken DNA and also between cDNA(gp) and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA(gp) did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.     

HPRS-103 (exogenous avian leukosis virus, subgroup J) has an env gene related to those of endogenous elements EAV-0 and E51 and an E element found previously only in sarcoma viruses.

The avian leukosis and sarcoma virus (ALSV) group comprises eight subgroups based on envelope properties. HPRS-103, an exogenous retrovirus recently isolated from meat-type chicken lines, is similar to the viruses of these subgroups in group antigen but differs from them in envelope properties and has been assigned to a new subgroup, J. HPRS-103 has a wide host range in birds, and unlike other nontransforming ALSVs which cause late-onset B-cell lymphomas, HPRS-103 causes late-onset myelocytomas. Analysis of the sequence of an infectious clone of the complete proviral genome indicates that HPRS-103 is a multiple recombinant of at least five ALSV sequences and one EAV (endogenous avian retroviral) sequence. The HPRS-103 env is most closely related to the env gene of the defective EAV-E51 but divergent from those of other ALSV subgroups. Probing of restriction digests of line 0 chicken genomic DNA has identified a novel group of endogenous sequences (EAV-HP) homologous to that of the HPRS-103 env gene but different from sequences homologous to EAV and E51. Unlike other replication-competent nontransforming ALSVs, HPRS-103 has an E element in its 3' noncoding region, as found in many transforming ALSVs. A deletion found in the HPRS-103 U3 EFII enhancer factor-binding site is also found in all replication-defective transforming ALSVs (including MC29, which causes rapid-onset myelocytomas).     

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.