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。     

SPF鸡胚中内源性白血病病毒全基因序列鉴定与分析

摘要:以国内某3家SPF鸡场的SPF鸡胚成纤维细胞提取的基因组DNA为模板,参照已发表的序列,设计合成了4对检测内源性白血病病毒引物,分别检测gag基因、pol基因、env基因和LTR片段,结果显示4者检出阳性率很高(gag,29/46;pol,27/46;env,24/46;LTR,31/46).设计合成了8对引物,选取4片段检测均为阳性的样品之一,经PCR成功扩增出了8段连续的、相互部分重叠的目的DNA片段,分别连接入T载体进行克隆测序.用DNAstar软件对测序结果进行拼接,从一个鸡胚得到了内源性白血病病毒前病毒全基因组序列.比较分析发现,该序列env基因与已知的E亚群内源性病毒代表株env基因的核苷酸序列同源性在98.5%以上,全基因组序列同源性在99.1%以上,而与其他亚群代表株同源性相对较低,env基因同源性仅为56.3%～91.5%.     

我国地方品种鸡分离到的一个禽白血病病毒新亚群的鉴定

为探明我国地方品种鸡群禽白血病病毒(Avian leukosis virus,ALV)的特点,通过接种DF-1细胞及细胞培养上清液p27抗原的检测,从芦花鸡中分离得到三株外源性ALV禽白血病病毒,分别是JS11C1、JS11C2和JS11C3,并对其进行亚群鉴定分析。用PCR方法扩增env基因测序,并与已知鸡源各亚群ALV的囊膜蛋白(gp85)作氨基酸同源性比较。这三株ALV的env基因的gp85大小为1 005bp,编码335个氨基酸;env基因的gp37大小为609bp,编码203个氨基酸。三个毒株之间gp85的同源性为91.9%～97.0%。与A、B、C、D和E五个经典亚群在GenBank中已发表的18个毒株的gp85的同源性仅在77.7%～84.6%间,显著低于鸡群中常见的A、B、E各亚群内的同源性范围(分别为88.2%～98.5%,91.6%～98.8%和97.9%～99.4%),而与J亚群参考株的同源性更是只有34.2%～36.5%。上述结果表明,芦花鸡分离到的三株病毒可能是不同于鸡源ALV已知6个亚群的一个新亚群,按国际上对ALV亚群分类的习惯,初步将其定名为K亚群。     

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。     

J亚群禽白血病病毒蛋鸡分离株SD07LK1全基因组核苷酸序列的比较

[目的]分析我国ALV-J蛋鸡分离株的来源和进一步演变趋势.[方法]以J亚群禽白血病病毒(ALV-J)蛋鸡分离株SD07LK1感染的鸡胚成纤维细胞(CEF)基因组:DNA作为其前病毒基因组模板,根据已发表序列设计合成9对引物,经PCR扩增出9段连续的、相互部分重叠的DNA片段和闭合环形前病毒两末端LTR的连接区段,并分别连入T载体进行克隆、测序.[结果]用:DNAstar软件对测序结果进行剪辑和拼接,首次完成了ALV-J蛋鸡分离株SD07LK1的前病毒全基因组核苷酸序列.[结论]将该序列与另外已完成的全基因组序列的比较表明,ALV-J的整个基因组gag和pol基因相对保守,各毒株间对应基因的同源性分别在95.O%以上,env基因的同源性仅为88.6%～94.0%.     

J亚群禽白血病病毒蛋鸡分离株SD07LK1全基因组核苷酸序列的比较分析

[目的]分析我国ALV-J蛋鸡分离株的来源和进一步演变趋势.[方法]以J亚群禽白血病病毒(ALV-J)蛋鸡分离株SD07LK1感染的鸡胚成纤维细胞(CEF)基因组:DNA作为其前病毒基因组模板,根据已发表序列设计合成9对引物,经PCR扩增出9段连续的、相互部分重叠的DNA片段和闭合环形前病毒两末端LTR的连接区段,并分别连入T载体进行克隆、测序.[结果]用:DNAstar软件对测序结果进行剪辑和拼接,首次完成了ALV-J蛋鸡分离株SD07LK1的前病毒全基因组核苷酸序列.[结论]将该序列与另外已完成的全基因组序列的比较表明,ALV-J的整个基因组gag和pol基因相对保守,各毒株间对应基因的同源性分别在95.O%以上,env基因的同源性仅为88.6%～94.0%.     

鸡传染性支气管炎病毒变异株全基因组序列分析

为了明确传染性性支气管炎病毒(Infectious bronchitis virus,IBV)分离株CK/CH/SD09/005的分子特征,以进一步丰富国内IBV的分子流行病学信息。本研究设计了25对引物对其全基因组进行了序列测定,并与参考株进行了同源性比较和S1基因遗传进化分析。结果显示CK/CH/SD09/005基因组为27 691bp(不包括5′端Cap和3′端Poly A)。全基因组同源性比对发现,CK/CH/SD09/005仅与GenBank中广西2009年分离株GX-NN09032各基因高度同源(97%~99%)。除GX-NN09032外,CK/CH/SD09/005基因组5′端复制酶基因(Gene 1)和3′端非转录区(Untranslated region,UTR)与2个QX基因型参考株ck/CH/LDL/091022和SDIB821/2012同源性最高,分别为97%和98%,但是3′端结构蛋白和非结构蛋白基因(S-3a-3b-3c/E-M-5a-5b-N)与这两个毒株同源性较低,仅为72%~90%。其ORF3c/E、5a、5b和N分别与韩国分离株1011、国内分离株CK/CH/LXJ/02I、DK/CH/HN/ZZ2004和YX10同源性最高,分别为97%、96%、99%和96%,而其ORF3a、3b和M与参考株同源性均低于90%。S1基因遗传进化分析发现,CK/CH/SD09/005和国内外39个参考株形成7个进化分支(基因型),CK/CH/SD09/005和2007以来几个分离株属于基因Ⅳ型,与其它6个基因型参考株S1和S2基因同源性为66%~69%和72%~81%,S1基因不仅表现广泛性点突变,而且有多处碱基插入和缺失,S2仅表现点突变。本研究结果表明CK/CH/SD09/005是一个变异株,可能是QX基因型IBV流行株与其它毒株重组进化而来,此外还涉及基因突变、插入和缺失等多种变异机制。     

绵羊肺腺瘤病毒内蒙古分离株前病毒全基因组克隆及序列分析北大核心CSCD

为了探究绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)内蒙古流行株与国际各代表株间的亲缘关系,本研究以内蒙古地区绵羊肺腺瘤病自然病例的肺组织总DNA为模板,克隆gag、pro与pol基因,并应用双酶切的方法将其与本实验室先期制备并保存的LTR、env基因连接起来,得到了含有JSRV内蒙古分离株前病毒全基因组重组质粒pMD-JSRV。序列分析结果表明,JSRV内蒙古分离株前病毒全基因组全长7 690bp,具外源性JSRV典型的分子特征:1在gag基因中含Sca I酶切位点;2核衣壳蛋白区有2个保守的可形成锌指结构的"CCHC"基序;3env基因编码的TM区胞浆尾部包含保守的特异性"YXXM"基序。将其进行同源性分析,结果表明该株病毒属JSRV-II型,与分离自美国的代表株AF105220间亲缘关系较近,同源性达95%。本研究为进一步探讨JSRV内蒙古分离株基因组结构特点与其致病机制间的关系奠定了基础。     

绵羊肺腺瘤病毒内蒙古分离株前病毒全基因组克隆及序列分析

为了探究绵羊肺腺瘤病毒(Jaagsiekte sheep retrovirus,JSRV)内蒙古流行株与国际各代表株间的亲缘关系,本研究以内蒙古地区绵羊肺腺瘤病自然病例的肺组织总DNA为模板,克隆gag、pro与pol基因,并应用双酶切的方法将其与本实验室先期制备并保存的LTR、env基因连接起来,得到了含有JSRV内蒙古分离株前病毒全基因组重组质粒pMD-JSRV。序列分析结果表明,JSRV内蒙古分离株前病毒全基因组全长7 690bp,具外源性JSRV典型的分子特征:1在gag基因中含Sca I酶切位点;2核衣壳蛋白区有2个保守的可形成锌指结构的"CCHC"基序;3env基因编码的TM区胞浆尾部包含保守的特异性"YXXM"基序。将其进行同源性分析,结果表明该株病毒属JSRV-II型,与分离自美国的代表株AF105220间亲缘关系较近,同源性达95%。本研究为进一步探讨JSRV内蒙古分离株基因组结构特点与其致病机制间的关系奠定了基础。     

植物活性长末端重复序列反转录转座子研究进展

长末端重复序列(Long terminal repeat,LTR)反转录转座子是真核生物基因组中普遍存在的一类可移动的DNA序列,它们以RNA为媒介,通过"复制粘贴"机制在基因组中不断自我复制。在高等植物中,许多活性的LTR反转录转座子已被详尽研究并应用于分子标记技术、基因标签、插入型突变及基因功能等分析。本文对植物活性LTR反转录转座子进行全面的调查,并对其结构、拷贝数和分布以及转座特性进行系统的归纳,分析了植物活性LTR反转录转座子的gag(种属特异抗原)和pol(聚合酶)序列特征,以及LTR序列中顺式调控元件的分布。研究发现自主有活性的LTR反转录转座子必须具备LTR区域以及编码Gag、Pr、Int、Rt和Rh蛋白的基因区。其中两端LTR区域具有高度同源性且富含顺式调控元件;Rt蛋白必备RVT结构域;Rh蛋白必备RNase_H1_RT结构域。这些结果为后续植物活性LTR反转录转座子的鉴定和功能分析奠定了重要基础。     

Isolation and identification of a subgroup A avian leukosis virus from imported meat-type grand-parent chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%–90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for ALV of subgroup A isolated from imported breeders.     

Isolation and Identification of a Subgroup A Avian Leukosis Virus from Imported Meat-type Grand-parent Chickens

An exogenous avian leukosis virus (ALV) strain SDAU09C1 was isolated in DF-1 cells from one of 240 imported 1-day-old white meat-type grand parent breeder chicks. Inoculation of SDAU09C1 in ALV-free chickens induced antibody reactions specific to subgroup A or B. But gp85 amino acid sequence comparisons indicated that SDAU09C1 fell into subgroup A; it had homology of 88.8%-90.3% to 6 reference strains of subgroup A, much higher compared to other subgroups including subgroup B. This is the first report for A...     

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。     

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.     

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。     

Soluble forms of the subgroup A avian leukosis virus [ALV(A)] receptor Tva significantly inhibit ALV(A) infection in vitro and in vivo

The interactions between the subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and soluble forms of the ALV(A) receptor Tva were analyzed both in vitro and in vivo by quantitating the ability of the soluble Tva proteins to inhibit ALV(A) entry into susceptible cells. Two soluble Tva proteins were tested: the 83-amino-acid Tva extracellular region fused to two epitope tags (sTva) or fused to the constant region of the mouse immunoglobulin G heavy chain (sTva-mIgG). Replication-competent ALV-based retroviral vectors with subgroup B or C env were used to deliver and express the two soluble tv-a (stva) genes in avian cells. In vitro, chicken embryo fibroblasts or DF-1 cells expressing sTva or sTva-mIgG proteins were much more resistant to infection by ALV(A) ( approximately 200-fold) than were control cells infected by only the vector. The antiviral effect was specific for ALV(A), which is consistent with a receptor interference mechanism. The antiviral effect of sTva-mIgG was positively correlated with the amount of sTva-mIgG protein. In vivo, the stva genes were delivered and expressed in line 0 chicken embryos by the ALV(B)-based vector RCASBP(B). Viremic chickens expressed relatively high levels of stva and stva-mIgG RNA in a broad range of tissues. High levels of sTva-mIgG protein were detected in the sera of chickens infected with RCASBP(B)stva-mIgG. Viremic chickens infected with RCASBP(B) alone, RCASBP(B)stva, or RCASBP(B)stva-mIgG were challenged separately with ALV(A) and ALV(C). Both sTva and sTva-mIgG significantly inhibited infection by ALV(A) (95 and 100% respectively) but had no measurable effect on ALV(C) infection. The results of this study indicate that a soluble receptor can effectively block infection of at least some retroviruses and demonstrates the utility of the ALV experimental system in characterizing the mechanism(s) of viral entry.     

Identification and characterization of a shared TNFR-related receptor for subgroup B, D, and E avian leukosis viruses reveal cysteine residues required specifically for subgroup E viral entry

Genetic and receptor interference data have indicated the presence of one or more cellular receptors for subgroup B, D, and E avian leukosis viruses (ALV) encoded by the s1 allele of the chicken tvb locus. Despite the prediction that these viruses use the same receptor, they exhibit a nonreciprocal receptor interference pattern: ALV-B and ALV-D can interfere with infection by all three viral subgroups, but ALV-E only interferes with infection by subgroup E viruses. We identified a tvb(s1) cDNA clone which encodes a tumor necrosis factor receptor-related receptor for ALV-B, -D, and -E. The nonreciprocal receptor interference pattern was reconstituted in transfected human 293 cells by coexpressing the cloned receptor with the envelope (Env) proteins of either ALV-B or ALV-E. This pattern of interference was also observed when soluble ALV surface (SU)-immunoglobulin fusion proteins were bound to this cellular receptor before viral challenge. These data demonstrate that viral Env-receptor interactions can account for the nonreciprocal interference between ALV subgroups B, D, and E. Furthermore, they indicate that a single chicken gene located at tvb(s1) encodes receptors for these three viral subgroups. The TVB(S1) protein differs exclusively at residue 62 from the published subgroup B- and D-specific receptor, encoded by the s3 allele of tvb. Residue 62 is a cysteine in TVB(S1) but is a serine in TVB(S3), giving TVB(S1) an even number of cysteines in the extracellular domain. We present evidence for a disulfide bond requirement in TVB(S1) for ALV-E infection but not for ALV-B infection. Thus, ALV-B and ALV-E interact in fundamentally different ways with this shared receptor, a finding that may account for the observed biological differences between these two ALV subgroups.     

系统发生分析发现牛病毒性腹泻病病毒新基因亚型

本研究对我国首次分离获得的牛源牛病毒性腹泻病毒(BVDV)毒株Changchun 184(CC-184)和猪源牛病毒 性腹泻病毒ZM-95进行了遗传衍化关系研究。选择主要抗原E2基因为研究对象,首先应用RT-PCR及套式PCR 克隆得到CC-184和ZM-95的E2片段,通过序列测定发现CC-184和ZM-95 E2基因长度分别为1,122bp和1, 125bp,各自编码374和375个氨基酸残基。核酸序列同源性比较和系统发生分析表明2株病毒均属于BVDV-1, CC-184与Osloss亲缘关系最近,都属于已有的b基因亚型,其E2基因同源性达91．8％。而ZM-95的E2基因有 一个特征性的变异区,包含一个密码子序列插入,这一变异区编码了一段有别于其他瘟病毒的五肽氨基酸序列 HYKKK。结果还表明ZM-95与BVDV-1现有的5个基因亚型的亲缘关系均较远,E2基因同源性最高(与Oregon c24v)只有72．4％。而BVDV-1亚型内毒株间的同源性大于85％,亚型间的同源性在69％～75％之间,充分说明 ZM-95是BVDV-1中一个新发现的基因亚型。通常认为猪源BVDV来源于牛,应该与牛源BVDV有十分近的遗 传关系,但是本研究发现ZM-95与其他已知牛源BVDV较低的基因同源性说明猪源BVDV还具有独立的遗传衍 化与传播来源的可能性。