A Recombinant Avian Leukosis Virus Subgroup J for Directly Monitoring Viral Infection and the Selection of Neutralizing Antibodies

Avian leukosis virus subgroup J (ALV-J) has induced serious clinical outbreaks and has become a serious infectious disease of chickens in China. We describe here the creation of a recombinant ALV-J tagged with the enhanced green fluorescent protein (named rHPRS-103EGFP). We successfully utilize the rHPRS-103EGFP to visualize viral infection and for development of a simplified serum-neutralization test.     

gga-miR-375 Plays a Key Role in Tumorigenesis Post Subgroup J Avian Leukosis Virus Infection

Avian leukosis is a neoplastic disease caused in part by subgroup J avian leukosis virus J (ALV-J). Micro ribonucleic acids (miRNAs) play pivotal oncogenic and tumour-suppressor roles in tumour development and progression. However, little is known about the potential role of miRNAs in avian leukosis tumours. We have found a novel tumour-suppressor miRNA, gga-miR-375, associated with avian leukosis tumorigenesis by miRNA microarray in a previous report. We have also previously studied the biological function of gga-miR-375; Overexpression of gga-miR-375 significantly inhibited DF-1 cell proliferation, and significantly reduced the expression of yes-associated protein 1 (YAP1) by repressing the activity of a luciferase reporter carrying the 3′-untranslated region of YAP1. This indicates that gga-miR-375 is frequently downregulated in avian leukosis by inhibiting cell proliferation through YAP1 oncogene targeting. Overexpression of gga-miR-375 markedly promoted serum starvation induced apoptosis, and there may be the reason why the tumour cycle is so long in the infected chickens. In vivo assays, gga-miR-375 was significantly downregulated in chicken livers 20 days after infection with ALV-J, and YAP1 was significantly upregulated 20 days after ALV-J infection (P<0.05). We also found that expression of cyclin E, an important regulator of cell cycle progression, was significantly upregulated (P<0.05). Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is related to caspase-dependent apoptosis, was also significantly upregulated after infection. Our data suggests that gga-miR-375 may function as a tumour suppressor thereby regulating cancer cell proliferation and it plays a key role in avian leukosis tumorigenesis.     

Infectivity of Proviral DNA from Avian Sarcoma Virus-Transformed Mammalian Cells

The number of Rous viral genomes in the cellular DNA from two subclones (RS2/3, RS2/6) derived from the same clone of hamster BHK-21 cells transformed by Rous sarcoma virus was determined by hybridization with viral complementary DNA made in vitro, and the capacity of the cellular DNA to infect (transfect) chicken embryo fibroblasts was compared before and after shearing this DNA to about the size of the provirus (6 x 10(6) to 7 x 10(6) daltons). The two subclones differed widely both in their capacity to give rise to virus (inducibility) after fusion with chicken embryo fibroblasts and in level of expression of viral proteins. It was shown that cells of both subclones contain a single copy of Rous DNA and yield infectious DNA. However, whereas transfection of chicken embryo fibroblasts was successful with both unsheared (>/=18 x 10(6) daltons) and sheared DNA from the most inducible subclone (RS2/3 subclone), which also expresses viral proteins to an appreciable amount, transfection with DNA from the least inducible subclone (RS2/6 subclone), in which viral proteins are not expressed, succeeded only with sheared DNA. It was then about as successful as with sheared or unsheared RS2/3 DNA. The lack of infectivity of unsheared RS2/6 DNA may be explained by the hypothesis proposed by Cooper and Temin (G. M. Cooper and H. T. Temin, J. Virol. 17:422-430, 1976) to explain the lack of infectivity of DNA from certain chicken cells producing spontaneously low amounts of RAV-0 and resistant to exogenous RAV-0 infection, that is, that the viral genome (proviral DNA) is linked to a cis-acting control element which blocks its expression. This linkage might originate, in RS2/6 cells, from translocation of cellular DNA containing the single proviral copy.     

Component of Strain MC29 Avian Leukosis Virus with the Property of Defectiveness

Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.     

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。     

抗J亚群禽白血病病毒的鸡胚成纤维细胞系建立

将J亚群禽白血病病毒囊膜基因(ALV-Jenv)插入pcDNA3.1真核表达载体,构建成转移载体pcDNA-env,并转染鸡胚成纤维细胞系DF-1,通过Zeocin药物筛选获得转染阳性细胞。细胞经传30代后,冻存。13个月后复苏,置不含药物的培养基中传代培养。连续传40代后,分别用PCR、Southern blot、间接免疫荧光(IFA)及Western blot对该细胞中的env基因进行了检测,并进行了抗病毒感染分析。研究结果表明,通过Zeocin药物筛选获得了稳定表达ALV-Jenv基因的鸡胚成纤维细胞系,命名为pcDNA-env-DF-1细胞。该细胞在体外抗ALV-J感染试验中,能抵抗1×104个TCID50病毒感染量,提示所构建的细胞系具有良好的抗病毒作用。这一细胞系的构建为研究ALV-J Env蛋白与宿主细胞表面受体相互作用分子机理提供了很好的工具。     

Characterization of Determinants for Envelope Binding and Infection in Tva, the Subgroup A Avian Sarcoma and Leukosis Virus Receptor

Tva is the cellular receptor for subgroup A avian leukosis and sarcoma virus (ALSV-A). The viral interaction domain of Tva is determined by a 40-residue, cysteine-rich module closely related to the ligand binding domain of the human low-density lipoprotein receptor (LDLR). In this report, we examined the role of the LDLR-like module of Tva in envelope binding and viral infection by mutational analysis. We found that the entire LDLR module in Tva is essential for efficient binding to the viral envelope protein. However, the 17 N-terminal residues of this module can be deleted without affecting receptor function, suggesting that the major determinants for viral entry are located at the C terminus of the module. The effect on viral infection of many amino acid substitutions and deletions in the LDLR module is context dependent, suggesting that the residues important for viral entry are dispersed throughout the LDLR module. In addition, we found that all 27 mutations at residues D46, E47, and W48 greatly reduced envelope binding. These results are discussed in relation to a recently elucidated structure for an LDLR module.     

蛋鸡J亚群禽白血病的分子生物学诊断

根据J亚群白血病病毒(ALV-J)原型株HPRS-103的序列设计了一对针对外源性ALV-J引物H5和H7,从发生ML病死鸡的肿瘤、骨髓、肝脏、脾脏和输卵管组织中提取DNA作为模板,经PCR扩增得到长度为545bp的片段,对其序列进行测定后,与ALV-J原型株HPRS-103的序列进行了比较,发现其核苷酸同源性为97．4％,所编码氨基酸的同源性为96．1％。该片段含有ALV-J gp85编码基因的部分序列和ALV-J pol基因的部分序列,从分子水平上证实了蛋鸡发生J亚群禽白血病,进一步证明了此前根据病理学观察、免疫组化及免疫荧光诊断的结果。这是首次从分子水平上证明蛋用型鸡发生J亚群禽白血病。     

Differential Proteome Analysis of Chikungunya Virus Infection on Host Cells

Background

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.

Methodology and Principal Findings

The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE). Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP) and cell cycle regulation.

Conclusion/Significance

This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1) regulation (in favour of virus survival, replication and transmission). While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.     

Cell Cycle-Dependent Activation of Rous Sarcoma Virus-Infected Stationary Chicken Cells: Avian Leukosis Virus Group-Specific Antigens and Ribonucleic Acid

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary cells did not contain avian leukosis virus group-specific antigens or ribonucleic acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV endogenous RNA-directed DNA polymerase activity.     

禽白血病病毒p19基因末端片段在大肠杆菌中的表达

根据禽白血病病毒（ALV）p19基因末端序列合成一条82bp的双链DNA片段,将其克隆到表达质粒pGE-MEX-1中,序列分析结果与设计的相符。重组表达质粒转化的大肠杆菌BL21（ＤＥ３）经ＩＰＴＧ诱导后产生３４kD融合表达产物,与理论值相符;Western-blot分析表明该表达产物能与兔抗ＡＬＶ血清发生反应。     

禽白血病病毒p19基因末端片段在大肠杆菌中的表达

根据禽白血病病毒（ALV）p19基因末端序列合成一条82 bp的双链DNA片段,将其克隆到表达质粒pGEMEX-1中,序列分析结果与设计的相符。重组表达质粒转化的大肠杆菌BL21（DE3）经IPTG诱导后产生34kD融合表达产物,与理论值相符;Western-blot分析表明该表达产物能与兔抗ALV血清发生反应。     

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     

黄羽肉鸡J亚群白血病病毒的分子生物学特性和致病性

通过接种鸡胚成纤维细胞(CEF)、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),首次从中国地方品系-黄羽肉鸡中分离到J亚群白血病病毒(ALV-J),并对其gp85基因和3′Ter序列及其致病性作了比较分析。结果显示,GD0510A、GD0510B和GD0512的gp85基因编码的氨基酸序列与国内毒株HN0001同源性最高,分别为94.1%、92.5%和95.8%,GD0510A和GD0512的3′Ter核酸序列与国内毒株同源性比较高。分离到的两株ALV-J(GD0510A和GD0512)感染1日龄肉鸡后出现明显生长抑制(P<0.05),并诱发中枢免疫器官法氏囊和胸腺萎缩。两株病毒单独感染均能降低鸡体对新城疫病毒和禽流感病毒(AIV-H5)疫苗的抗体滴度,GD0512感染鸡后能明显抑制感染鸡对新城疫病毒疫苗的免疫反应(P<0.05),而GD0510A感染鸡后在4w时也能明显抑制感染鸡对禽流感病毒(AIV-H5)疫苗的免疫反应(P<0.05)。研究证实在我国地方品系黄羽肉鸡存在ALV-J的感染,分子生物学特性研究表明该毒株可能是来自白羽肉鸡且能造成感染鸡的免疫抑制。     

Polymorphism of Avian Leukosis Virus Subgroup E Loci Showing Selective Footprints in Chicken

Avian leukosis virus subgroup E (ALVE) is a family of endogenous retroviruses in the chicken genome. To investigate the genetic consequences of chicken domestication, we analyzed 18 ALVE loci in red jungle fowls, layers, broilers, and Chinese indigenous chickens. None of the ALVE loci tested were found in red jungle fowls, but 12 were present in domestic chickens. ALVE1 and ALVE16 are found in regions of the genome that harbor quantitative trait loci (QTL) affecting egg production traits. ALVE1 was fixed and ALVE16 was detected only in layers. By contrast, ALVE-b1, ALVE-b5, ALVE-b6, and ALVE-b8 integrated into regions of the genome that harbor QTL affecting meat production traits. Carrier frequencies of these four ALVE loci were high in broilers and low in Chinese local chickens; the loci were not found in the layers. This study demonstrated that insertionally polymorphic ALVE loci can illustrate the selective footprints in the chicken genome.     

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。