新疆伊犁地区驴脑组织亨德拉病毒感染情况调查

目的调查新疆伊犁地区草原放养驴群中亨德拉病毒(Hendra virus,HeV)中枢感染的流行情况。方法采用一步法实时荧光定量RT-PCR检测病毒核酸片段的方法对采自新疆伊犁地区草原放养、未接种HeV疫苗的65例驴脑组织进行HeV核蛋白(N)基因片段检测。结果65例驴脑组织成功进行一步法实时荧光定量RT-PCR检测,未检出阳性样本。结论目前尚未发现我国新疆伊犁地区放养驴中存在HeV中枢神经感染的证据,提示该地区出现HeV感染流行的可能性不大。     

新疆部分地区动物Nipah病毒和Hendra病毒感染的调查

拟建立一步法实时RT-PCR检测Nipah病毒(Nipah virus,NiV)、Hendra病毒(Hendra virus,HeV)的方法,对采集自中国新疆部分地区动物外周血样本进行NiV和HeV检测,以获得我国新疆地区的NiV和HeV的病毒流行病学资料.采用NiV和HeV一步法实时RT-PCR检测方法,对2007年9-12月采集的新疆伊犁河谷地区以及巴音布鲁克草原放养的500匹马、100匹驴和160只犬外周血单个核细胞RNA进行NiV和HeV N基因片段检测.结果:对采集的动物血样本进行NiV和HeV核酸检测,成功进行了一步法实时RT-PCR反应,没有发现阳性样本.初步流行病学研究提示我国新疆部分地区天然牧场放养的动物中可能不存在NiV和HeV感染,该地区出现NiV和HeV爆发的可能性较小.     

新疆伊犁地区马脑组织西尼罗病毒感染的调查

目的了解新疆伊犁地区草原放养马群中西尼罗病毒(W estN ile virus,WNV)中枢感染的流行状况。方法采用一步法实时荧光定量逆转录聚合酶链反应(Real Tim e RT-PCR)对采自新疆伊犁地区草原放养、未接种WNV疫苗的189例马脑组织进行WNV包膜蛋白(E)基因片段检测。结果被检马脑组织标本中未发现WNV E基因片段。结论目前尚没有证据表明我国新疆伊犁地区的放养马中存在WNV脑炎的感染,提示该地区出现WNV脑炎流行的可能性小。     

中国学术期刊网络出版总库节点文献新疆驴脑组织尼帕病毒N基因片段的检测

目的检测新疆伊犁草原地区放养新疆驴的脑组织样本中尼帕病毒（Nipah Virus,NiV）核蛋白（N）基因片段,调查该地区放养新疆驴NiV感染流行状况。方法采用一步法实时荧光定量逆转录聚合酶链反应（one-step Real-Time FQ RT-PCR）对采自新疆伊犁地区草原放养且未接种NiV疫苗的65例新疆驴脑组织进行NiVN基因片段检测。结果新疆驴脑组织标本中未检出NiV N基因片段。结论目前尚无证据表明我国新疆伊犁地区新疆驴中存在NiV感染,提示该地区短时间内爆发该病毒感染可能性较小。     

新疆驴脑组织尼帕病毒N基因片段的检测

目的检测新疆伊犁草原地区放养新疆驴的脑组织样本中尼帕病毒(Nipah Virus,NiV)核蛋白(N)基因片段,调查该地区放养新疆驴NiV感染流行状况。方法采用一步法实时荧光定量逆转录聚合酶链反应(one-step Real-Time FQ RT-PCR)对采自新疆伊犁地区草原放养且未接种NiV疫苗的65例新疆驴脑组织进行NiVN基因片段检测。结果新疆驴脑组织标本中未检出NiV N基因片段。结论目前尚无证据表明我国新疆伊犁地区新疆驴中存在NiV感染,提示该地区短时间内爆发该病毒感染可能性较小。     

巢式荧光定量RT-PCR检测中枢神经系统HCV方法的构建

目的 建立丙型肝炎病毒5′NCR基因的巢式荧光定量RT-PCR检测方法 ,用于中枢神经系统感染超低浓度HCV的准确和快速定量检测.方法 选择高度保守区5′NCR基因片段,设计并合成相应的特异性引物和探针,建立巢式荧光定量RT-PCR检测中枢神经系统感染样本中的超低浓度HCV正负链RNA的方法 .并对32例血清HCV抗体检查阴性的病毒性脑炎患者脑脊液有核细胞和外周血单个核细胞(PBMC)进行检测.结果 可特异性地检出脑脊液有核细胞及PBMC中HCV正负链RNA,最低检出浓度均可达7.85 copies/μl,与其他单股正链RNA病毒如登革热病毒(DEV)无交叉反应.32例血清HCV抗体检查阴性样本脑脊液有核细胞和PBMC中 HCV 5′NCR正链片段阳性的分别为1例(1/32)和2例(2/32),负链片段阳性的分别为1例(1/32)和0例(0/32).结论 本方法 的构建适用于中枢神经系统感染超低浓度HCV正负链RNA检测,且快速有效、敏感性和特异性较高,不易出现假阳性,可有效提高检出率,进一步完善目前临床常规检测HCV的方法 .     

灭活贝氏柯克斯体免疫小鼠抗登革病毒感染的实验研究

目的 研究灭活贝氏柯克斯体增强小鼠非特异性抗登革病毒感染的免疫效能。方法 用灭活贝氏柯克斯体全细胞疫苗(WCV)免疫BALB/c小鼠,每只小鼠皮下注射50μg WCV,初次免疫后第5周及第7周分别腹腔注射20μg WCV加强免疫。1周后,用10~5 TCID_(50)剂量的登革病毒2型经尾静脉注入感染小鼠,于感染后48、72h分别解剖小鼠.自血和脑组织提取RNA样本,用实时荧光定量聚合酶链反应(PCR)检测样本。结果 用登革病毒特异的定量PCR检测感染小鼠血RNA样本,结果显示感染72h血样本中病毒含量显著低于48h样本,但免疫小鼠与未免疫小鼠之间无显著差异。检测脑组织RNA样本,未免疫小鼠和免疫小鼠的感染48h样本检出病毒均为少量;但未免疫小鼠感染96h样本检出病毒量明显增加,显著高于免疫小鼠。结论 灭活贝氏柯克斯体可诱导机体产生非特异的免疫应答,具有一定增强小鼠抗登革病毒感染的能力,但具体机制有待进一步研究。     

琉球病毒、索尔韦齐病毒、苏里斯病毒等三种沙粒病毒实时荧光RT-PCR检测方法研究

建立可检测琉球病毒(Ryukyu virus,RYKV),索尔韦齐病毒(Solwezi virus,SOLV)以及苏里斯病毒(Souris virus,SOUV)等三种沙粒病毒的实时荧光定量RT-PCR检测方法.分析三种病毒流行病学分布特征,从国际公共数据库搜索下载基因组序列,进行比对分析,确定检测靶标,借助primer premier 6.0生物信息学软件,设计引物和探针,建立实时荧光定量RT-PCR检测方法,利用化学合成和体外转录方法制备模拟样本,比较评价三种方法的检测限、特异性、重复性特征.所建实时荧光定量RT-PCR检测方法均可有效扩增检测病毒RNA靶标,检测限分别为40拷贝/μL、7拷贝/μL和15拷贝/μL,检测汉城病毒、汉滩病毒、登革病毒Ⅰ～Ⅳ型分离株、发热伴血小板减少综合病毒及30份健康人血清样本无非特异性扩增,三种病毒相互间以及其他8种出血热相关沙粒病毒RNA间无交叉反应,重复性比较分析显示变异系数均在2％以内.本研究建立的检测RYKV、SOLV和SOUV三种沙粒病毒的实时荧光RT-PCR方法具备用于相关疑似感染者临床样本、宿主动物标本以及进出口物品的筛查检测的潜力,但因未经基于实际病毒感染样本的比较评价,检测结果的解释仍具有一定的局限性.     

Hendra病毒一步法Real-time RT-PCR检测方法的建立

目的 建立针对Hendra病毒N基因的一步法Real-time RT-PCR检测方法,用于Hendra病毒感染样本的快速检测和准确定量.方法 针对Hendra病毒的保守基因N设计引物和探针,构建体外转录的RNA片段作为标准品,建立一步法Real-time RT-PCR反应方法并分析敏感性和特异性.结果 所设计的引物经Blast检索可以用于检测所有已知的Hendra病毒株.本研究建立的一步法Real-time RT-PCR方法可以特异性检测出Hendra病毒,不与Nipah病毒产生交叉反应.检测灵敏度为2.6×100～2.6×101copies/μl.标准曲线的线性范围为2.6×101～2.6×107copies/μl.结论 本研究建立的一步法Real-time RT-PCR方法敏感性和特异性较高,且不易出现污染引起的假阳性结果,适合用于Hendra病毒感染样本的检测.     

呼吸道合胞病毒TaqMan探针实时定量RT-PCR检测方法的建立及应用

目的:建立呼吸道合胞病毒(RSV)核酸特异、快速、敏感的TaqMan探针实时荧光定量PCR检测方法,并对临床样本进行检测。方法:比对编码RSV非编码蛋白的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量RT-PCR检测方法,并与传统RT-PCR方法进行比较,分别对两者的灵敏性、特异性、重复性及临床样本检验的适用性进行评价。结果:所建立的实时荧光定量RT-PCR检测方法可用于RSV的特异性检测。相对于传统RT-PCR方法100拷贝/反应的检测灵敏度,实时荧光定量RT-PCR的检测灵敏度达到10拷贝/反应,检测范围为1010~101拷贝/反应,且具有良好的特异性和重复性。从169份临床呼吸道标本中检出RSV阳性40例,高于普通PCR方法(31/169)。结论:建立了RSV的TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,在临床上具有较好的应用前景。     

Routes of Hendra Virus Excretion in Naturally-Infected Flying-Foxes: Implications for Viral Transmission and Spillover Risk

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.     

Spatiotemporal Aspects of Hendra Virus Infection in Pteropid Bats (Flying-Foxes) in Eastern Australia

Hendra virus (HeV) causes highly lethal disease in horses and humans in the eastern Australian states of Queensland (QLD) and New South Wales (NSW), with multiple equine cases now reported on an annual basis. Infection and excretion dynamics in pteropid bats (flying-foxes), the recognised natural reservoir, are incompletely understood. We sought to identify key spatial and temporal factors associated with excretion in flying-foxes over a 2300 km latitudinal gradient from northern QLD to southern NSW which encompassed all known equine case locations. The aim was to strengthen knowledge of Hendra virus ecology in flying-foxes to improve spillover risk prediction and exposure risk mitigation strategies, and thus better protect horses and humans. Monthly pooled urine samples were collected from under roosting flying-foxes over a three-year period and screened for HeV RNA by quantitative RT-PCR. A generalised linear model was employed to investigate spatiotemporal associations with HeV detection in 13,968 samples from 27 roosts. There was a non-linear relationship between mean HeV excretion prevalence and five latitudinal regions, with excretion moderate in northern and central QLD, highest in southern QLD/northern NSW, moderate in central NSW, and negligible in southern NSW. Highest HeV positivity occurred where black or spectacled flying-foxes were present; nil or very low positivity rates occurred in exclusive grey-headed flying-fox roosts. Similarly, little red flying-foxes are evidently not a significant source of virus, as their periodic extreme increase in numbers at some roosts was not associated with any concurrent increase in HeV detection. There was a consistent, strong winter seasonality to excretion in the southern QLD/northern NSW and central NSW regions. This new information allows risk management strategies to be refined and targeted, mindful of the potential for spatial risk profiles to shift over time with changes in flying-fox species distribution.     

Flying-Fox Roost Disturbance and Hendra Virus Spillover Risk

Bats of the genus Pteropus (flying-foxes) are the natural host of Hendra virus (HeV) which periodically causes fatal disease in horses and humans in Australia. The increased urban presence of flying-foxes often provokes negative community sentiments because of reduced social amenity and concerns of HeV exposure risk, and has resulted in calls for the dispersal of urban flying-fox roosts. However, it has been hypothesised that disturbance of urban roosts may result in a stress-mediated increase in HeV infection in flying-foxes, and an increased spillover risk. We sought to examine the impact of roost modification and dispersal on HeV infection dynamics and cortisol concentration dynamics in flying-foxes. The data were analysed in generalised linear mixed models using restricted maximum likelihood (REML). The difference in mean HeV prevalence in samples collected before (4.9%), during (4.7%) and after (3.4%) roost disturbance was small and non-significant (P = 0.440). Similarly, the difference in mean urine specific gravity-corrected urinary cortisol concentrations was small and non-significant (before = 22.71 ng/mL, during = 27.17, after = 18.39) (P= 0.550). We did find an underlying association between cortisol concentration and season, and cortisol concentration and region, suggesting that other (plausibly biological or environmental) variables play a role in cortisol concentration dynamics. The effect of roost disturbance on cortisol concentration approached statistical significance for region, suggesting that the relationship is not fixed, and plausibly reflecting the nature and timing of disturbance. We also found a small positive statistical association between HeV excretion status and urinary cortisol concentration. Finally, we found that the level of flying-fox distress associated with roost disturbance reflected the nature and timing of the activity, highlighting the need for a ‘best practice’ approach to dispersal or roost modification activities. The findings usefully inform public discussion and policy development in relation to Hendra virus and flying-fox management.