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。     

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     

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。     

鸡内源性类J亚群禽白血病病毒gp85基因的克隆及分析

为深入探讨J亚群禽白血病病毒(Avian leukosis virus subgroup J,ALV-J)的亚群特性,利用ALV-J gp85基因两侧的序列片段为引物,从正常SPF蛋鸡、商品肉鸡和DF1细胞基因组中完整地扩增了内源性类ALV-J gp85基因。肉鸡和DF1细胞内源性类ALV-J gp85基因同源性达99．9％;SPF蛋鸡内源性类ALV-J gp85基因与肉鸡和DF1细胞的内源性类ALV-J gp85基因之间同源性达95．6％、95．3％。三种不同来源的内源性类ALV-J gp85基因DNA与IMC10200株ALV-J的gp85基因的同源性分别为91．8％、94．1％、94．0％;与ALV-J原型株HPRS-103gp85基因的同源性分别为95．6％、98．3％、99．9％。内源性类ALV-J gp85序列与外源性ALV-J gp85基因具有相似或一致的ORF和Jameson-Worrlf抗原表位优势。     

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.     

两个猪瘟病毒野毒株gp55基因抗原编码区序列的分析及比较

利用反转录－PCR方法扩增了吉林省猪瘟病毒（HCV）两个野毒株gp55基因的主要保护性抗原编码区,并将其克隆到pGEM－T载体中,然后用Sanger双脱氧法测定了其核苷酸序列,并推导了其氨基酸序列。将测定的这两个HCV野毒株的部分序列（350bp）与国内外已知的HCV序列进行比较,结果表明：这两个野毒株的核苷酸序列的同源性为94．9％,氨基酸序列同源性为97．4％,与1985～1992年意大利中部分离4个野毒株的同源性明显高于其它HCV毒株,核苷酸同源性分别为97．2％～98．3％和94.0％～949％,氨基酸同源性分别为98．3％～991％和97.4％～98.3％,而与我国的HCV标准强毒株即石门株的核苷酸同源性仅分别为83.1％和83.1％,氨基酸同源性仅分别为90.6％和91.4％。因此认为吉林省这两个野毒株与意大利中部的4个野毒株具有密切的关系,而与石门株很可能来源不同。     

我国部分地区H9亚型禽流感病毒血凝素基因序列比较与遗传发生关系分析

为从分子水平掌握我国H9亚型AIV的遗传变异情况和流行规律,本研究汇集近年来从我国12个省、市、自治区的发病鸡群中分离到的23株H9亚型禽流感病毒,通过RT-PCR方法和核苷酸序列测定获得了23个毒株的HA基因cDNA核苷酸序列。核苷酸和推导的氨基酸序列同源性比较结果表明,这些毒株HA基因的核苷酸序列同源性为94．1％～100％,氨基酸序列同源性为95．4％～100%;将这23个毒株和来自亚洲及世界其它地区的另外31株的HA基因cDNA序列同源性进行比较发现,分离自香港的HK170499株与日本的2个毒株关系较近;氨基酸序列分析发现,CKGS199、CKTJ196、CKTJ296、CKSH300和CKBJ197五个毒株各发生了一个潜在的糖基化位点的丢失。54株H9亚型AIVHA基因55bp～1152bp的氨基酸序列分析发现,裂解位点尽管有10种基序,但本研究中的23株和近年来从我国大陆和香港地区的分离的毒株则均为RSSR↓GLF;构成受体结合位点的191位氨基酸有一个规律,即所有中国大陆毒株与部分香港毒株都为N,其它毒株均为H,141aa～143aa处的糖基化位点有与191aa类似的规律,即：凡是191aa为N的毒株,该处均为NVS（CKBJ194除外）,凡是191aa为H的毒株,则该处均为NVT;遗传发生关系分析,中国大陆毒株处于欧亚谱系的第一支。本研究结果表明近年来我国鸡群中H9N2亚型禽流感病毒的感染流行可能有一个共同的来源,这为制定防治该亚型禽流感流行的有效对策提供了重要的科学依据。     

犬瘟热病毒TN株血凝基因的克隆与序列分析

用RT PCR方法对分离的TN野毒株H基因进行了扩增 ,并将其克隆到pGEM T载体上 ,进行核苷酸序列测定。结果TN野毒株H基因ORF全长 1 ,81 5bp ,编码 60 4个氨基酸。与GenBank中己报道的 7个CDV毒株相比 ,H基因核苷酸序列的同源性在 92 %～ 99%之间 ,推导的H蛋白氨基酸序列的同源性在 91 %～ 99%之间。TN株H蛋白潜在的N 联糖基化位点为 8个 ,而Onderstepoort弱毒株H蛋白为 4个 ;其中N端第 1 9～ 2 1、 30 9～ 31 1、 5 8     

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).     

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.     

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).     

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.     

The avian retrovirus env gene family: molecular analysis of host range and antigenic variants.

The nucleotide sequence of the env gp85-coding domain from two avian sarcoma and leukosis retrovirus isolates was determined to identify host range and antigenic determinants. The predicted amino acid sequence of gp85 from a subgroup D virus isolate of the Schmidt-Ruppin strain of Rous sarcoma virus was compared with the previously reported sequences of subgroup A, B, C, and E avian sarcoma and leukosis retroviruses. Subgroup D viruses are closely related to the subgroup B viruses but have an extended host range that includes the ability to penetrate certain mammalian cells. There are 27 amino acid differences shared between the subgroup D sequence and three subgroup B sequences. At 16 of these sites, the subgroup D sequence is identical to the sequence of one or more of the other subgroup viruses (A, C, and E). The remaining 11 sites are specific to subgroup D and show some clustering in the two large variable regions that are thought to be major determinants of host range. Biological analysis of recombinant viruses containing a dominant selectable marker confirmed the role of the gp85-coding domain in determining the host range of the subgroup D virus in the infection of mammalian cells. We also compared the sequence of the gp85-coding domain from two subgroup A viruses, Rous-associated virus type 1 and a subgroup A virus of the Schmidt-Ruppin strain of Rous sarcoma virus. The comparison revealed 24 nonconservative amino acid changes, of which 6 result in changes in potential glycosylation sites. The positions of 10 amino acid differences are coincident with the positions of 10 differences found between two subgroup B virus env gene sequences. These 10 sites identify seven domains in the sequence which may constitute determinants of type-specific antigenicity. Using a molecular recombinant, we demonstrated that type-specific neutralization of two subgroup A viruses was associated with the gp85-coding domain of the virus.     

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...     

Artificial insertion of a dominant gene for resistance to avian leukosis virus into the germ line of the chicken

Summary This report describes the unique biological properties of a transgenic chicken line that contains a defective avian leukosis virus (ALV) proviral insert that we call alv6. Chick embryo fibroblasts (CEF) containing this insert express subgroup A envelope glycoprotein since they yield focus-forming pseudotype virus when co-cultivated with transformed quail cells expressing envelope-defective Bryan high-liter Rous sarcoma virus (RSV). In addition, these cells display high interference to subgroup A RSV but not to subgroup B RSV infection. Chickens containing this insert are highly resistant to pathogenic subgroup A ALV infection, but show little immunological tolerance to subgroup B ALV infection. Thus we have artificially inserted a dominant gene for resistance to avian leukosis infection into the chicken germ line.