替换HIV-1衣壳蛋白基因SHIV的构建及其活性测定

将猴免疫缺陷病毒(Simianimmunodeficiencyvirus,SIVmm239)中gag基因的衣壳蛋白部分置换成人免疫缺陷病毒(Humanimmunodeficiencyvirustype1,HIV-1HXBc2)的相应部分,构建出替换了衣壳蛋白基因的人/猿嵌合免疫缺陷病毒(SHIV)原病毒DNA。用此SHIV原病毒DNA转染293T细胞,细胞中能够检测到嵌合病毒基因的转录与翻译;在细胞培养液上清中亦可检测到装配出的病毒颗粒。病毒颗粒形态正常,含有基因组RNA,具有反转录酶活性,嵌合的外源衣壳蛋白能够正确剪切,形成棒状的核心。将此嵌合SHIV病毒感染MT4细胞,病毒能够吸附并进入细胞,能完成反转录过程,但不能增殖。     

牛免疫缺陷病毒反式激活因子功能域的研究

牛免疫缺陷病毒(Bovine immunodeficiency virus, BIV)与人免疫缺陷病毒(Human immunod eficiency virus, HIV)同属反转录病毒科慢病毒属[1].BIV基因组5′端的长末端重复序列(LTR)起始病毒结构基因和非结构基因的转录[2],因而许多细胞因子和病毒编码的调节蛋白作用于LTR,以调节BIV的基因表达.     

人类免疫缺陷病毒解剖学病毒储存库与检测方法的研究进展

由于人类免疫缺陷病毒(human immunodeficiency virus,HIV)储存库的存在,获得性免疫缺陷综合征(acquired immunodeficiency syndrome,AIDS)患者即便接受高效抗反转录病毒治疗也无法完全清除体内的潜伏病毒.本文就HIV在人体内可能存在的解剖学储存库、病毒储存库...     

BIV在人源细胞MT-4中的活性研究

牛免疫缺陷病毒(Bovine immunodeficiency virus, BIV)在分类上属于反转录病毒科的慢病毒属,目前尚未见BIV感染人的报道.为进一步确定BIV对人源细胞的感染性,我们用BIV127cDNA转染人源细胞MT-4,通过RT-PCR检测到BIVgag基因的转录,IFA则显示BIV127的gag或gag-pol基因在MT-4细胞中得到了翻译,而RT值的测定也有力地说明BIV127cDNA已经在MT-4细胞内表达出有活性的反转录酶,但细胞传代实验表明BIV不能在MT-4细胞内复制.     

猪瘟病毒衣壳蛋白靶向核酸酶表达系统的建立及鉴定

根据猪瘟病毒衣壳蛋白(C)基因序列设计一对引物,RT-PCR扩增获得编码猪瘟病毒衣壳蛋白的C基因,将其插入到含有葡萄球菌核酸酶(SN)基因的真核表达载体pcDNA-SN中,筛选获得重组质粒pcDNA-C-SN。脂质体转染猪肾细胞(PK-15),并经G418稳定筛选,通过RT-PCR、免疫印迹和间接免疫荧光鉴定表达的融合蛋白,体外DNA消化试验检测核酸酶活性。结果表明融合蛋白C-SN在PK-15细胞中获得了稳定表达,能够被兔抗猪瘟病毒衣壳蛋白多抗所识别,并具有良好的核酸酶活性,能够对DNA进行切割。同时,稳定表达融合蛋白C-SN的PK-15细胞系中能够有效抑制猪瘟野毒的增殖,使其感染性降低102～103倍。这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于抵抗猪瘟病毒感染奠定了基础。     

嵌合人/牛免疫缺陷病毒cDNA(pHBIV-2)传染性克隆的构建及其生物学活性

为探讨牛免疫缺陷病毒(BIV)Tat能否在功能上取代HIV Tat,构建用BIV tat取代HIV tat的嵌合人/牛免疫缺陷病毒(pHBIV-2)cDNA,将其转染人源MT4细胞.PCR、RT-PCR法检测到嵌合基因组在MT4细胞中可稳定地存在并转录;套式Alu-PCR法检测到嵌合基因组可整合到细胞基因组中;RTase活性测定及IFA检测显示,嵌合基因在MT4细胞中得到了翻译.结果表明,HIV的tat基因用BIVtat取代后产生的传染性cDNA克隆,仍能在人源MT4细胞中产生有复制性的重组病毒.     

牛泡沫病毒长末端重复序列在大肠杆菌中的启动子功能

泡沫病毒具有复杂的基因组结构,包含典型反转录病毒共有的长末端重复序列(long terminal repeat,LTR)启动子,负责起始结构基因gag、pol、env的表达,并在env基因内部存在独有的内部启动子(internal promoter,IP)[13],用以起始下游反式作用因子Tas(在BFV中称Borf-1)的表达.有报道劳斯肉瘤病毒(rous sarcoma virus,RSV)和人免疫缺陷病毒(human immunodeficiency virus,HIV)LTR可在E.coli中起始基因表达[4,7].泡沫病毒至今未见报道.     

克隆的口蹄疫病毒蛋白疫苗——在牛和猪体内的反应

从口蹄疫病毒A_(12)的病毒粒子RNA中制取得到编码其致免疫的衣壳蛋白VP_3的DNA序列,并把它接到质粒上,通过大肠杆菌色氨酸起动—操纵系统表达出一个嵌合蛋白。当被这个质粒转化的大肠杆菌在无色氨酸培养基上生长时,全部细胞蛋白中有大约17％是不溶性的,稳定嵌合蛋白。纯化的嵌合蛋白与口蹄疫病毒的VP_3蛋白等克分子地竞争抗口蹄病毒的特异性抗体。给六头牛、二头猪接种这种蛋白能诱发出高水平的中和抗体和抗口蹄疫病毒侵袭的保护反应。     

患病中国大鲵中分离到一株虹彩病毒及其特性的研究

从陕西某大鲵养殖场患病的大鲵体内分离到一株病毒。患病大鲵以体表溃疡,特别是肢体远端溃烂为主要临床特征。该病毒于10℃~30℃能在BF-2(Caudal trunk cells of blue-gillfry)、CO(Gorad cells of grass carp)、CHSE(Embryo cells of Chinook salmon)、FHM(cells of fathed minnow)等细胞中较好地增殖,最适生长温度为25℃~30℃。病毒对氯仿、热、pH3、pH10敏感,DNA抑制剂5-氟-2′-脱氧尿苷(5-fluro-2-′deoxyuridine,FUDR)能抑制病毒在细胞中的增殖,提示该病毒是有囊膜的DNA病毒。经电镜观察,在感染了病毒的细胞切片中可见到大量直径约130~150 nm有囊膜的六角形病毒颗粒成晶格排列在细胞质里,病毒呈典型的虹彩病毒形态。抽提病毒核酸后进行PCR扩增,用已知蛙病毒主要衣壳蛋白(MCP)基因的保守序列设计的引物能扩增出431bp的片段。扩增的片段测序后,和已知的几种蛙病毒属成员的主要衣壳蛋白基因中的相应片段进行比对,相似性在96%以上。血清学试验结果显示该病毒和IPNV(Infectious pancreatic necrosis virus,IPNV)、GCRV(Grass carp reovirus,GCRV)、SVCV(Spring viraemia of carp virus,SVCV)I、HNV(Infectious hematopoietic necrosis virus,IHNV)在血清学上没有相关性。以上结果提示该病毒可能是虹彩病毒科蛙病毒属的成员,暂时命名为大鲵虹彩病毒(Andrias davidianus iridovirus,ADIV)。该病毒与大鲵发病的关系有待进一步研究。     

HIV储存库特征及其主要检测方法

获得性免疫缺陷综合症(acquired immunodeficiency syndrome,AIDS),即艾滋病,是由人类免疫缺陷病毒(human immunodeficiency virus,HIV)引起的一种致死率极高的慢性传染病。如何有效控制和预防AIDS已成为全球共同面临的严峻挑战。HIV DNA整合在CD4+T细胞基因组中形成HIV储存库,使部分HIV逃避了宿主免疫清除和抗病毒药物的治疗作用。如何彻底清除患者体内HIV储存库成为功能性治愈艾滋病的重大障碍。了解HIV储存库的病毒学特征及其检测方法,是目前HIV研究的热点之一。现对HIV储存库特征及其主要检测方法作一综述。     

猴免疫缺陷病毒（SIV）实时荧光定量PCR检测方法的建立

目的建立快速、敏感、特异的猴免疫缺陷病毒（SIV）TaqMan探针实时荧光定量PCR检测方法,对SIV病毒核酸进行定量检测。方法RT—PCR扩增SIVmac251保守gag基因序列796bp片段,进行TA克隆,构建标准品质粒pMD—SIVgag。通过对SIV定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量PCR方法的灵敏度、特异性和重复性。结果所建立的SIVQPCR检测方法,质粒DNA模板在10’～10。拷贝之间表现较好线性和相关性,标准曲线所得斜率为-3．26,相关系数为0．999。检测灵敏度达到200拷贝,方法重复性测试,检测25份临床样品CV％均小于1％。结论建立的SIVQPCR检测方法特异性、敏感性高,稳定性好,可用于定量测定猴免疫缺陷病毒（SIV）核酸拷贝量。     

Bovine alpha-2-HS-glycoprotein functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and SIVmac239 envelope glycoprotein

The presence of anti-CCR5 and anti-HIV-1 envelope glycoprotein (ENV) gp41 antibodies (Abs) at sites of HIV-1 exposure was effective in preventing its transmission to HIV-1-exposed seronegative (ESN) subjects. Here, we design an immunogen that can induce Abs against CCR5 and SIVmac239 ENV simultaneously and show that bovine alpha-2-HS-glycoprotein (bAHSG) functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and ENV. Initially, we generated a rhesus CCR5-derived cyclopeptide (cDDR5) conjugated with a recombinant trimeric SIVmac239 Env. When inguinally administered to rhesus macaques, the immunogen simultaneously induced both anti-CCR5 and anti-ENV Abs in sera, and the purified serum IgG fraction exerted an inhibitory effect on SIVmac239 infection in vitro. When further boosted with bAHSG, the responses of both Abs were significantly enhanced. To examine the cross-reactivity of bAHSG, it was administered to naïve cynomolgus macaques. The results showed a statistically significant increase in IgG response against cynomolgus CCR5 and SIVmac239 ENV, and the induction of neutralizing activity against SIVmac239. These findings suggest that bAHSG is useful for immune strategies aimed at generating Abs against CCR5 and ENV simultaneously to confer HIV-protective immunity.     

Rates of amino acid change in the envelope protein correlate with pathogenicity of primate lentiviruses

A spectrum of pathogenicity has been observed for primate lentiviruses in their natural hosts. For example, human immunodeficiency virus type 1 (HIV-1) is a potent etiologic agent for AIDS in man, whereas there is no evidence to date which indicates that simian immunodeficiency virus from African green monkeys (SIVAGM) causes immunodeficiency in AGM. We measured the relative rates of amino acid change, as the ratio of the number of nonsynonymous to synonymous (silent) nucleotide substitutions, for six primate lentiviruses evolving in their respective hosts. These rates for the external envelope glycoprotein (gp120) and gag coding sequences are 2–3 times higher for pathogenic HIV-1 and SIV..ac (macaque) than for minimally pathogenic SIVAGM and SIVsn,m (sooty mangabey), and intermediate for HIV-2. We speculate that the increased rates of nonsynonymous changes in gp120 and gag coding sequences are due to viral escape from immune surveillance and are indicative of higher immunogenicity of these proteins in their hosts. Based on these results and available experimental data, we conclude that there is a positive correlation between lentiviral pathogenicity and immunogenicity of the Env and Gag proteins in a given host. This hypothesis is consistent with recent data suggesting that immune system activation or autoimmunity induced by viral antigens may be important in the pathogenesis of AIDS.Correspondence to: E.G. Shpaer     

SHIV感染恒河猴十二指肠黏膜活检组织病理学观察

目的使用消化内窥镜对SHIV感染恒河猴十二指肠黏膜进行观察。方法SHIV感染恒河猴,消化内镜检查并进行活检取材,对活检标本进行固定,石蜡包埋,常规HE染色镜检。结果内镜大体观察胃黏膜和十二指肠黏膜基本上完整平滑,1例胃黏膜有弥漫性白斑,3例有小面积的出血;光镜下十二指肠黏膜完整未见脱落,固有层中有散在分布的慢性炎细胞,活检取材最深可达黏膜下层,未见其他特异性病理改变。结论不同毒株,不同感染时间的SHIV感染恒河猴和对照恒河猴的十二指肠黏膜活检组织HE染色组织结构清晰,呈慢性炎症改变,病理形态上没有明显的差异。     

Experimental Infection of African Green Monkeys and Cynomolgus Monkeys With a SIVAGM Strain Isolated From a Healthy African Green Monkey

An infection occurred in all African green monkeys and cynomolgus monkeys experimentally inoculated with SIV_AGM [TYO-1], as demonstrated by the appearance of an antibody to SIV_AGM [TYO-1] and the isolation of the virus. No monkey exhibited overt clinical disorders throughout the experimental period of 42 weeks. Thus, SIV_AGM was not pathogenic to its original host or to macaques, This system is proposed as a model for HIV infection manifesting no overt disease.     

Manipulating the Interferon Signaling Pathway: Implications for HIV Infection

During human immunodeficiency virus(HIV) infection, type I interferon(IFN-I) signaling induces an antiviral state that includes the production of restriction factors that inhibit virus replication, thereby limiting the infection. As seen in other viral infections, type I IFN can also increase systemic immune activation which, in HIV disease, is one of the strongest predictors of disease progression to acquired immune deficiency syndrome(AIDS) and non-AIDS morbidity and mortality.Moreover, IFN-I is associated with CD4 T cell depletion and attenuation of antigen-specific T cell responses. Therefore,therapeutic manipulation of IFN-I signaling to improve HIV disease outcome is a source of much interest and debate in thefield. Recent studies have highlighted the importance of timing(acute vs. chronic infection) and have suggested that specific targeting of type I IFNs and their subtypes may help harness the beneficial roles of the IFN-I system while avoiding its deleterious activities.     

The V1 region of gp120 is preferentially selected during SIV/HIV transmission and is indispensable for envelope function and virus infection

A transmission bottleneck occurs during each human immunodeficiency virus(HIV) transmission event, which allows only a few viruses to establish new infection. However, the genetic characteristics of the transmitted viruses that are preferentially selected have not been fully elucidated. Here, we analyzed amino acids changes in the envelope protein during simian immunodeficiency virus(SIV)/HIV deep transmission history and current HIV evolution within the last 15–20 years. Our results confirmed that the V1V2 region of gp120 protein, particularly V1, was preferentially selected. A shorter V1 region was preferred during transmission history, while during epidemic, HIV may evolve to an expanded V1 region gradually and thus escape immune recognition. We then constructed different HIV-1 V1 mutants using different HIV-1 subtypes to elucidate the role of the V1 region in envelope function. We found that the V1 region, although highly variable, was indispensable for virus entry and infection, probably because V1 deletion mutants exhibited impaired processing of gp160 into mature gp120 and gp41. Additionally, the V1 region affected Env incorporation. These results indicated that the V1 region played a critical role in HIV transmission and infection.     

人中性粒细胞多肽HNP1,3体外抗单纯疱疹病毒Ⅰ型的作用

体外观察人中性粒细胞多肽1,3(Human neutrophil peptide,HNP1,3)及阿昔洛韦(Acyclovir,ACV)对单纯疱疹病毒Ⅰ型(Herpes simplex virus 1,HSV-1)的抑制作用.以Vero细胞为靶细胞,用各种浓度HN1,3与游离病毒颗粒(直接失活组)及感染病毒后的靶细胞(复制抑制组)进行相互作用,镜下观察各药物对HSV-1致细胞病变效应的抑制作用,并采用ELISA法测定感染48h后药物对HSV-1囊膜糖蛋白分泌的抑制作用.MTT法检测各药物对细胞的毒性作用.结果显示直接失活组中,HNP1,3可使HSV-1的致细胞病变效应减轻,对HSV-1直接失活的50%有效浓度(ECs0)为8.1μg/mL、10.03μg/mL;复制抑制组中,ACV使HSV-1的致细胞病变效应减轻,EC5o为0.68μg/mL.MTT检测结果表明HNP1,3在治疗浓度范围内无明显细胞毒性.以上结果表明HNP1,3除具有较强的抗菌作用和抗人类免疫缺陷病毒Ⅰ型(Human immunodeficiency virus 1,HIV-1)活性外,还能失活HSV-1病毒颗粒,从而逆转病毒及其蛋白的病毒效应(致细胞病变)和抑制病毒蛋白质的合成.