2003-2008年我国实验猴BV抗体检测结果及抗体水平变化规律的分析

目的对近年来我国实验猴BV（猴疱疹病毒Ⅰ型）抗体检测结果进行比较分析,以了解我国实验猴BV感染情况及其抗体水平变化规律,为我国实验猴质量控制及标准化提供依据。方法根据国标中ELISA方法 ,对2003～2008年我国11个单位送检的2个品种猴血清进行BV抗体检测,并对检测结果进行统计分析。结果检测的4612份猴血清中,有1843份BV抗体呈阳性,阳性率为39.96%;6年中检测猴群BV抗体阳性率基本在30%～50%。幼年（≤2岁）、青年（2.1～4.0岁）、成年（4.1～6.5岁）、老年（≥6.5岁）4个不同年龄段猴BV感染率分别为26.28%、31.53%、53.74%、87.27%。不同年龄感染率差异显著（P〈0.01）。雌猴BV感染率（35.91%）高于雄猴（34.93%）,但两者差异不显著（P〉0.05）。结论不同年龄猴BV感染率不同,随着年龄的增长BV感染率升高。     

安徽恒河猴的微生物学和寄生虫学检测

对安徽省实验猕猴中心的安徽恒河猴进行了微生物（包括病毒和病原菌）和寄生虫检测。对恒河猴的病毒检测结果发现,猕猴疱疹病毒1型（BV）和猴痘病毒（SPV）抗体的阳性率分别为20．7％（6／29）和10．0％（2／20）,20只恒河猴中没有发现猴反转录D型病毒（SRV）、猴免疫缺陷病毒（SIV）和猴T细胞趋向性病毒Ⅰ型（STLV—1）的抗体。5只受检的人工繁育的安徽恒河猴没有感染沙门菌、皮肤病原真菌、志贺菌和结核分枝杆菌的这四种病原菌。肉眼检测恒河猴体表,未发现体外寄生虫。39份人工繁殖的恒河猴粪便样品的总寄生虫感染率为38．5％,检测到溶组织内阿米巴和5种蠕虫（粪类圆线虫、猴结节线虫、绦虫、钩虫、蛔虫）,感染率最高的是粪类圆线虫和猴结节线虫。本次调查表明,安徽恒河猴无特殊疾病,健康状况基本良好,可以建立普通级的实验恒河猴,实现安徽恒河猴的实验动物化。     

安徽野生和自繁恒河猴血液生化指标的测定与分析

目的测定人工饲养条件下安徽野生和自繁恒河猴的血液生化指标,并比较分析两种来源的恒河猴,雌、雄猴间以及感染BV阳性与阴性恒河猴生化指标的差异性。方法采用全自动生化分析仪对安徽野生和自繁恒河猴的14个血液生化指标进行测定,并用统计学方法比较了相同性别的野生猴与自繁猴以及感染BV阳性与阴性恒河猴血液生化值的差异性。结果野生猴与自繁猴雄性的生化指标普遍高于雌性,野生猴碱性磷酸酶、甘油三脂和谷氨酰基转移酶雌雄间差异显著;自繁猴碱性磷酸酶、白蛋白、血清Ca、甘油三脂、肌酐和谷氨酰基转移酶雌雄间差异有显著性。除谷草转氨酶、尿素氮和血清总胆固醇外,感染BV阳性较感染BV阴性的恒河猴所得生化指标高。结论野生猴与自繁猴,雌雄间猴以及感染BV阳性与阴性猴的血液生化指标有一定的差异性。     

安徽野生和自繁恒河猴血液生化指标的测定与分析简

目的 测定人工饲养条件下安徽野生和自繁恒河猴的血液生化指标,并比较分析两种来源的恒河猴,雌、雄猴间以及感染BV阳性与阴性恒河猴生化指标的差异性.方法采用全自动生化分析仪对安徽野生和自繁恒河猴的14个血液生化指标进行测定,并用统计学方法比较了相同性别的野生猴与自繁猴以及感染BV阳性与阴性恒河猴血液生化值的差异性.结果 野生猴与自繁猴雄性的生化指标普遍高于雌性,野生猴碱性磷酸酶、甘油三脂和谷氨酰基转移酶雌雄间差异显著;自繁猴碱性磷酸酶、白蛋白、血清Ca、甘油三脂、肌酐和谷氨酰基转移酶雌雄间差异有显著性.除谷草转氨酶、尿素氮和血清总胆固醇外,感染BV阳性较感染BV阴性的恒河猴所得生化指标高.结论 野生猴与自繁猴,雌雄间猴以及感染BV阳性与阴性猴的血液生化指标有一定的差异性.     

恒河猴感染SARS-CoV的病毒学、血清学检测

目的对感染SARS-CoV的8只恒河猴进行病毒学、血清学指标检测。方法SARS-CoV经鼻腔接种8只恒河猴,在感染的第1天开始到5、7、10、15、20、30和60天分别安乐处死时,不同时间取咽拭子、血液和脏器,进行病毒分离,RT-PCR检测和抗体测定。结果RT-PCR证实感染病毒检出时间为5~16d,8只猴中的5只分离到了病毒,感染15d后可检测到抗体。结论感染SARS-CoV的恒河猴不仅出现与SARS患者类似的临床和病理学改变,也在一定时期内排毒,出现特异免疫反应,这些指标均可作为药物筛选、疫苗评价等方面的重要参数。     

CXCR4嗜性SHIVKU-1病毒静脉感染中国恒河猴初步研究

目的了解SHIVKU一1静脉途径感染中国恒河猴的感染特点及进展规律。方法两只健康中国恒河猴,静脉感染SHIVKU-1病毒,定期采样检测血浆病毒载量、CD4＋／CD8＋比值、CD4＋T细胞绝对数变化和血清中抗SHIVKU-1特异性IgG抗体水平。多色流式技术分析外周血、腹股沟淋巴结和十二指肠粘膜固有层CD4＋T淋巴细胞记忆细胞亚群变化。结果两只实验猴成功感染SHIVKU-1病毒,一直到感染后3个月均保持稳定水平的病毒载量。外周血CD4＋T淋巴细胞下降明显,CD4＋／CD8＋T细胞比值严重倒置。CD4＋Tcm细胞比例在经历了感染早期的下降后,大幅升高,尤其是外周血和淋巴结。CD4＋Tem则在粘膜固有层中增加明显。结论SHIVKU．1静脉途径成功感染了中国恒河猴,为SHIV／中国恒河猴疾病及评价模型的建立奠定了良好的基础,为今后使用此模型评价抗病毒药物或疫苗提供了条件。     

SHIV-KB9感染中国恒河猴动物模型的建立

目的为了进一步确证SHIV-KB9感染中国恒河猴的病毒浓度范围,测试动物对病毒的适应性,明确该动物模型的可重复性。方法实验前采集猴血清并进行血清学检查。选出4只无SIV、STLV、SRV/D和B病毒感染的恒河猴,分别用10倍系列稀释的病毒液静脉感染实验猴,使用流氏细胞术、血常规、病毒分离、DNA-PCR和RT-PCR等方法确定实验猴是否被感染,以及感染后恒河猴体内病毒复制和免疫细胞损伤情况。结果实验猴的血浆病毒载量、病毒分离结果、CD4＋/CD8＋比值和CD4＋T细胞数等证实,4.8×105 copies/mL以上浓度的SHIV-KB9病毒液能成功感染中国恒河猴。结论本研究进一步明确了SHIV-KB9感染中国恒河猴的有效病毒浓度范围,确定了SHIV-KB9病毒感染中国恒河猴的病毒学、免疫学的测定指标,成功的建立了SHIV-KB9/中国恒河猴动物模型。     

恒河猴piwil4基因的鉴定与表达分析

为了研究人类近亲恒河猴中PIWI家族蛋白PIWIL4的结构和表达情况,文章首次利用同源比对和RT-PCR方法克隆了恒河猴piwil4基因,检测了其mRNA在恒河猴心脏、脑、结肠、附睾和睾丸5种组织中的表达情况,利用生物信息学的方法对恒河猴piwil4基因和人的PIWIL4(HIWI2)基因编码的蛋白产物进行了同源性分析和结构域分析,并进一步利用免疫组化的方法比较了PIWIL4蛋白在成人、成年恒河猴和性未成熟恒河猴睾丸组织中的表达分布。结果表明,恒河猴piwil4 mRNA在多组织中表达,恒河猴和人的PIWIL4蛋白的氨基酸序列同源性达97%以上,均含有PAZ和Piwi结构域,它们在两物种成年个体睾丸组织中空间分布一致,但在不同发育阶段恒河猴睾丸组织中的分布发生了改变,幼猴中PIWIL4蛋白主要表达于生精小管细胞的细胞核,在成年猴睾丸组织中则表达于各种细胞的胞浆中。上述结果提示,piwil4基因在人类和恒河猴精子发生过程中作用类似,PIWIL4蛋白在幼猴和成年猴睾丸组织中的表达差异提示它们在不同发育阶段功能的改变。     

中国恒河猴Mamu-A*01基因筛查及其对抗原肽p11C的特异性CTL反应

目的 筛查中国恒河猴Mamu-A*01基因,比较中国恒河猴和印度恒河猴的Mamu-A*01基因序列和功能是否相同.方法 PCR方法检测128只中国恒河猴,用特异性引物扩增Mamu-A*01基因,将PCR扩增后的产物克隆测序后与印度恒河猴的Mamu-A*01基因进行同源比对;酶联免疫斑点检测 (ELISPOT) 方法分别检测5只Mamu-A*01基因阳性和5只阴性恒河猴针对SIV、SHIV抗原肽p11C的特异性CTL反应.结果 共筛查出5 只Mamu-A*01基因阳性恒河猴 (3.91%),经测序分析后与印度恒河猴的同源性可达99.1 %.这5只均为SIV/SHIV感染恒河猴,其中四只SIV感染的猴的ELISPOT结果显示针对p11C的高频CTL反应,斑点数在500-1400/106 PBMCs之间,而另1只SHIV感染的恒河猴及5只阴性猴没有斑点出现.结论 中国恒河猴含有Mamu-A*01基因,基因频率有区域性差异,中国恒河猴的Mamu-A*01可提呈特异性抗原肽p11C.     

RT-SHIV感染中国恒河猴及体内传代

目的了解RT-SHIV感染中国恒河猴的感染特点,研究RT-SHIV在中国恒河猴中传代特点;建立RT-SHIV中国恒河猴动物模型,为评价HIV-1药物有效性提供动物平台。方法选择4只健康恒河猴,其中两只动物经上肢静脉感染RT-SHIV病毒,感染急性期采取外周血分离CD8-PBMC,扩增病毒,将新制备的病毒静脉感染另外两只中国恒河猴,通过监测血浆病毒载量,CD4＋/CD8＋比值,CD4＋T淋巴细胞和B淋巴细胞的绝对数,了解实验猴的感染状态,同时分析病毒RT基因变异情况。结果 4只动物均获得系统性感染,且传代动物急性期表现更为强烈,RT基因在感染和传代的过程中共观察到3个氨基酸的改变。结论本研究为RT-SHIV中国恒河猴模型的建立提供了基础信息。     

Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus

Cellular immune responses mediated by CD8+ lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.     

Validation of an enzyme-linked immunosorbent assay kit using herpesvirus papio 2 (HVP2) antigen for detection of herpesvirus simiae (B virus) infection in rhesus monkeys

Serologic testing for antibody to monkey B virus (BV) in macaque sera is problematic due to the biohazardous nature of BV antigens. Herpesvirus papio 2 (HVP2), a herpesvirus of baboons, is nonpathogenic to humans and is genetically and antigenically more closely related to BV than is human herpes simplex virus 1. This paper describes the results of our in-house laboratory that compared a BV antigen-based enzyme-linked immunosorbent assay (ELISA) by commercial testing laboratory and an HVP2-based ELISA in our laboratory by using 447 sera from 290 rhesus monkeys. The HVP2-based ELISA identified as positive 99.11% of the sera identified as BV-positive by the BV ELISA. The BV antigen-based ELISA identified as positive 98.21% of the sera identified as BV-positive by the HVP2-based ELISA. The HVP2 ELISA also identified two BV-negative and six BV-equivocal sera as positive. Both ELISAs identified the same 85 negative and three equivocal samples as negative and equivocal, respectively. The high degree of correlation (weighted kappa coefficient, 0.94) between the two tests indicates that the HVP2 ELISA is a sensitive and reliable assay for in-house testing of the BV status of rhesus monkeys.     

A natural asymptomatic herpes B virus infection in a colony of laboratory brown capuchin monkeys (Cebus apella)

Herpes B virus (BV) infection of macaques persists in the natural host, but is mainly asymptomatic. However, BV can cause fatal disease in humans and in several non-macaque species such as capuchin monkeys (Cebus apella). The BV infection described here in a colony of capuchin monkeys was persistent but asymptomatic. Initially the infection was detected serologically in five out of seven animals. However, using polymerase chain reaction (PCR) developed specifically for BV, we found the virus in all seven clinically healthy animals. It is probable that the infection was transferred from BV-infected macaques housed in different cages but in the same room for several years. We have no evidence to indicate that similar asymptomatic infections may occur in other New World species but the possibility should not be discounted. We recommend that the housing of capuchin monkeys in close proximity to macaques should be avoided and that greater caution should be used when handling capuchin monkeys and possibly other New World species that have been in contact with macaques. All may act as a source of BV infection in humans, hence routine, repeated testing of all primates is essential.     

Simian immunodeficiency virus-specific CD8+ lymphocyte response in acutely infected rhesus monkeys.

To assess the possible role of cytotoxic T lymphocytes (CTLs) in containing the spread of human immunodeficiency virus in acutely infected individuals, the temporal evolution of the virus-specific CD8+ lymphocyte response was defined in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. A brief period of SIVmac plasma antigenemia was seen 9 to 16 days following intravenous infection with SIVmac, ending as the absolute number of CD8+ peripheral blood lymphocytes (PBLs) increased. In a prospective assessment of the ability of CD8+ lymphocytes of these monkeys to suppress SIVmac replication in autologous PBLs, inhibitory activity was detected as early as 4 days, with a more pronounced effect 12 to 16 days following infection. SIVmac Gag- and Nef-specific CD8+ effector cell activities were demonstrable in PBLs of animals by 2 weeks following virus inoculation. In fact, SIVmac-specific CTL precursors were documented in the PBLs of rhesus monkeys 4 to 6 days after SIVmac infection. These studies indicate that AIDS virus-specific CD8+ CTLs are present in PBLs within days of infection and may play an important role in containing the early spread of virus.     

Infection of macaque monkeys with simian immunodeficiency virus from African green monkeys: virulence and activation of latent infection

The virulence of three isolates of simian immunodeficiency virus from African green monkeys (SIVagm) was studied in rhesus and pigtailed macaques. None of 15 rhesus monkeys and one of four pigtailed monkeys died from infection during the time they were studied (up to 33 months). SIVagm was only isolated from rhesus monkeys for up to 2 months after inoculation. However, when these animals were secondarily infected with Simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1), SIVagm was activated and isolated. Dual infection caused increased mortality.     

Detection of a unique genotype of monkey B virus (Cercopithecine herpesvirus 1) indigenous to native Japanese macaques (Macaca fuscata)

The Japanese macaque or snow monkey (Macaca fuscata) is an autochthonous monkey in Japan. It has long been assumed that the monkey population was not infected with Cercopithecine herpesvirus 1 (monkey B virus [BV]) since cases of human BV infection have never been reported in Japan. Although serologic testing of captive snow monkeys in Japan revealed antibodies to BV, it was thought that native Japanese macaques had either been infected with herpes simplex virus from humans or with BV from other imported macaque species. To clarify this issue, we performed polymerase chain reaction (PCR) analysis to amplify BV sequences from trigeminal ganglia of 30 Japanese macaque monkeys that were seropositive for BV. Sequences from two BV genes, UL27 (360 bp) and UL19 (1.0 Kbp), from 3 of 30 monkeys were amplified. Results of restriction fragment length polymorphism analysis and DNA sequencing of the fragments provided evidence that native Japanese macaques are infected with BV. Phylogenetic analysis indicated that these monkeys harbor their own genotype of BV that is different from other known BV genotypes, and provided additional evidence supporting the co-evolution of BV and macaques.     

Association of activating KIR copy number variation of NK cells with containment of SIV replication in rhesus monkeys

While the contribution of CD8? cytotoxic T lymphocytes to early containment of HIV-1 spread is well established, a role for NK cells in controlling HIV-1 replication during primary infection has been uncertain. The highly polymorphic family of KIR molecules expressed on NK cells can inhibit or activate these effector cells and might therefore modulate their activity against HIV-1-infected cells. In the present study, we investigated copy number variation in KIR3DH loci encoding the only activating KIR receptor family in rhesus monkeys and its effect on simian immunodeficiency virus (SIV) replication during primary infection in rhesus monkeys. We observed an association between copy numbers of KIR3DH genes and control of SIV replication in Mamu-A*01? rhesus monkeys that express restrictive TRIM5 alleles. These findings provide further evidence for an association between NK cells and the early containment of SIV replication, and underscore the potential importance of activating KIRs in stimulating NK cell responses to control SIV spread.