中国首次分离的Tahyna病毒毒株的生物学特性及分子进化特征分析

2006年夏季,本课题组在新疆喀什地区伽师县库蚊标本中分离到我国首株Tahyna病毒XJ0625株,并发现当地不明原因发热患者存在该病毒感染。本研究通过细胞培养、动物实验、电镜观察、间接免疫荧光及交叉保护中和试验等研究对XJ0625病毒株的细胞易感性、动物致病性、形态学及抗原性等特征进行观察,并应用分子生物学软件对其分子进化特征加以分析。结果发现该毒株可以引起BHK-21细胞病变,乳鼠颅内接种该毒株可以引起死亡。与其他布尼亚病毒形态相似,Tahyna病毒为球形有包膜病毒。该病毒与国际流行Tahyna病毒Bardos92株的抗体作用,在间接免疫荧光试验中呈现阳性荧光信号。空斑减少中和试验结果显示该抗体对XJ0625病毒株的中和效价为1∶3 200。核苷酸序列分析显示,该病毒与Bardos92株处于同一个进化分支,二者S节段同源性为91.8%,M节段同源性为81.9%。     

新型布尼亚病毒抗体检测方法的建立及初步应用简

目的：建立新型布尼亚病毒IgG抗体ELISA检测方法。方法：用基因工程重组表达的新型布尼亚病毒NP抗原包被酶联板,建立间接ELISA法检测新型布尼亚病毒IgG抗体,并进行特异性和灵敏度评价,健康人群中检测结果计算临界值（均值＋3标准差）。检测70例发热伴血小板减少综合征患者恢复血清和69份健康人血清样品。结果：在70份患者血清样品中,检测出新型布尼亚病毒IgG抗体阳性51例,阳性率为72．14％（51/70）;69份健康人血清样品中,检测出1份阳性,特异性为98．6％（1/69）。结论：建立的新型布尼亚病毒IgG抗体ELISA检测方法特异性强、灵敏度高,可用于新型布尼亚病毒感染的检测及流行病学调查。     

新型布尼亚病毒抗体检测方法的建立及初步应用

目的:建立新型布尼亚病毒IgG抗体ELISA检测方法。方法:用基因工程重组表达的新型布尼亚病毒NP抗原包被酶联板,建立间接ELISA法检测新型布尼亚病毒IgG抗体,并进行特异性和灵敏度评价,健康人群中检测结果计算临界值(均值+3标准差)。检测70例发热伴血小板减少综合征患者恢复血清和69份健康人血清样品。结果:在70份患者血清样品中,检测出新型布尼亚病毒IgG抗体阳性51例,阳性率为72.14%(51/70);69份健康人血清样品中,检测出1份阳性,特异性为98.6%(1/69)。结论:建立的新型布尼亚病毒IgG抗体ELISA检测方法特异性强、灵敏度高,可用于新型布尼亚病毒感染的检测及流行病学调查。     

发热伴血小板减少综合征患者血清内抗体IgG中和活性及其影响因素分析

发热伴血小板减少综合征(SFTS)的病原体为发热伴血小板减少综合征布尼亚病毒,目前尚无特异性防治措施。本研究旨在明确SFTS患者不同时期血清内抗体的中和活性,为将来开发单克隆中和抗体药物和疫苗设计提供可靠的血源。7名患者15份血清收集后采用protein A亲和纯化抗体,SDS-PAGE分析抗体的纯度和分子量大小,中和实验评估抗体的中和能力,结果发现7个患者15份血清中4个恢复期患者血清内抗体具有广谱中和能力,其余3个患者未产生中和抗体,所有患者急性期均未产生IgG中和抗体,产生中和抗体的个体内抗体的中和能力随病毒消长而发生改变。因此,机体感染后部分康复者建立了特异性体液免疫力。此外,SFTS患者年龄亦影响中和抗体的产生,高龄并不预示着免疫力逐渐丧失,推测个体差异决定了免疫力强弱。具有广谱中和能力的血源将为开发治疗SFTS的候选抗体药物以及疫苗设计提供可能。     

内蒙地区发热患者血清中冠状病毒抗体的检测

目的分析内蒙地区发热患者中冠状病毒的感染情况。方法以SARS冠状病毒感染Vero细胞涂片为冠状病毒抗原片,用间接免疫荧光法分别检测55例发热患者和68例正常人血清中冠状病毒的IgG、IgM抗体。结果发热患者血清中冠状病毒IgG抗体和IgM抗体阳性率分别为29.1%(16/55)和10.9%(6/55),而正常人血清中只检测到2.9%(2/68)的IgG抗体,且未检测到IgM抗体,2组患者的IgG和IgM抗体阳性率比较差异均有显著性;随机选取7例患者的IgG阳性血清进行SRAS冠状病毒的特异性抗体封闭实验,结果有6例血清仍为阳性,有1例血清转为阴性,说明冠状病毒IgG抗体阳性血清中85.7%为普通冠状病毒特异性,14.3%为SARS冠状病毒特异性。结论普通冠状病毒是内蒙地区发热患者的主要病原体之一,部分患者还存在SARS冠状病毒的既往感染。     

大学新生戊型肝炎病毒现/近期隐性感染的筛查

为了解大学新生戊型肝炎病毒(HEV)现/近期隐性感染的状况,探讨引起隐性感染与急性戊型肝炎(HE)的HEV毒株间有无差异,本实验利用ELISA一步法检测了正常大学新生血清标本中抗-HEV IgM,对抗-HEV IgM阳性者检测抗-HEV IgG,并进行HEV逆转录-巢式PCR(RT-nPCR). 结果2223份血清中抗-HEV IgM阳性18份,阳性率0.8%,P/N值在2.0~3.0之间.17份标本抗-HEV IgM、IgG同时阳性,1份抗-HEV IgM单独阳性,但用RT-nPCR检测抗-HEV IgM阳性标本, HEV RNA均阴性.提示正常大学新生中存在HEV现/近期隐性感染,应注意感染者作为传染源的可能性;HEV隐性感染时,产生的抗体滴度或亲和力较低,病毒血症时间短,病毒滴度低,或毒株的基因序列与引起急性HE的毒株的序列有一定差异.     

哈尔滨地区发热患者风疹病毒的分离鉴定

目的 为了明确哈尔滨地区风疹病毒（Rubella virus,RV）的流行情况,对2003年上半年采集的部分发热患者血清及血细胞进行筛选、鉴定、比较,进行血清学和病原学研究。同时从风疹病毒IgM抗体阳性患者血中分离病毒,对疑似风疹病毒的分离株进行鉴定。方法 采用酶联免疫吸附试验（ELISA）对159例发热患者血清中的风疹病毒IgM进行测定;采用BHK21细胞培养法对风疹病毒IgM抗体阳性患者血标本进行风疹病毒分离,并用RT—PCR、细胞生长曲线、电镜观察、免疫组化等方法对可疑风疹病毒株进行鉴定。结果 风疹病毒IgM抗体阳性为22例,占13．83％;经RT—PCR、细胞生长曲线、电镜观察、免疫组化等方法初步鉴定从血液标本中获得的病毒分离株是风疹病毒。结论 采用FTISA决对该地区159例发热患者血清风疹病毒IgM进行检测,阳性率为13．83％;获得了1株风疹病毒分离株。     

用捕捉ELISA法检测类脊髓灰质炎患者血清中肠道病毒71型IgM抗体以进行早期诊断

以肠道病毒71型(EV71)感染Vero细胞制备的EV71抗原,用于IgM捕捉ELISA法检测类脊髓灰质炎(类脊灰)患者血清中EV71 IgM抗体,特异性高,敏感性及稳定性良好。154例脊髓灰质炎(脊灰)病毒IgM阴性的可疑脊灰患者血清中EV71 IgM抗体阳性检出率为14.9％(23/154)。病后第2天的标本即可测出EV71 IgM,5至12天者抗体滴度较高,病后第40天的标本尚可测出EV71 IgM。本法是EV71感染的早期快速诊断方法,可用于与脊灰的鉴别诊断。     

流行性出血热病毒的蚀斑形成技术

国外已有报道,流行性出血热病毒(EHFV)能在Vero-E6细胞上形成蚀斑。由于蚀斑试验和蚀斑减少试验用于测定病毒滴度及中和抗体效价较其它方法准确,且特异性高,适用于测定出血热病毒间抗原性差异,感染或免疫后中和抗体水平。但由于EHFV在细胞内繁殖培养时间较长,蚀斑形成的细胞培养条件要求较高,目前国内尚未见成功的报道。我们基本     

大学新生戊型肝炎病毒现／近期隐性感染的筛查

为了解大学新生戊型肝炎病毒(HEV)现／近期隐性感染的状况,探讨引起隐性感染与急性戊型肝炎(HE)的HEV毒株间有无差异,本实验利用ELISA一步法检测了正常大学新生血清标本中抗—HEV IgM,对抗—HEV IgM阳性者检测抗—HEV IgG,并进行HEV逆转录一巢式PCR（RT-nPCR)。结果 2223份血清中抗—HEV IgM阳性18份,阳性率0．8％,P/N值在2．0—3．0之间。17份标本抗—HEV IgM、IgG同时阳性,1份抗—HEV IgM单独阳性,但用RT-nPCR检测抗—HEV IgM阳性标本,HEV RNA均阴性。提示正常大学新生中存在HEV现/近期隐性感染,应注意感染者作为传染源的可能性;HEV隐性感染时,产生的抗体滴度或亲和力较低,病毒血症时间短,病毒滴度低,或毒株的基因序列与引起急性HE的毒株的序列有一定差异。     

我国西尼罗病毒和Tahyna病毒的发现与流行

已发现100余种蚊传虫媒病毒在世界各地流行,其引发的人兽共患病是全世界关注的公共卫生问题。长期以来我国仅发现乙型脑炎和登革热两种蚊传虫媒病毒病,但近年来新发现西尼罗病毒和Tahyna病毒及其感染疾病流行。从我国新疆维吾尔自治区采集的蚊虫标本中分离到西尼罗病毒,大量血清学研究证明当地不仅存在西尼罗病毒感染所致疾病,还发生过西尼罗病毒感染引发的病毒性脑炎流行。目前已从新疆维吾尔自治区、青海省和内蒙古自治区采集的蚊虫标本中分离到Tahyna病毒,并发现其在自然界动物中的循环和导致的人类感染流行。西尼罗病毒和Tahyna病毒及其相关感染性疾病的发现为我国虫媒病毒及虫媒病毒病的预防与控制提出了新的挑战。     

The Neglected Arboviral Infections in Mainland China

The major arboviral diseases in mainland China include Japanese encephalitis, dengue fever, Crimean-Congo hemorrhagic fever (also known as Xinjiang hemorrhagic fever), and tick-borne encephalitis. These and other newly found arbovirus infections due to Banna virus and Tahyna virus contribute to a large and relatively neglected disease burden in China. Here we briefly review the literature regarding these arboviral infections in mainland China with emphasis on their epidemiology, primary vectors, phylogenetic associations, and the prevention programs associated with these agents in China.     

Development of a Flow-Trough Microarray based Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Assay for the Detection of European Bunyaviruses

It is suspected that apart from tick-borne encephalitis virus several additional European Arboviruses such as the sandfly borne Toscana virus, sandfly fever Sicilian virus and sandfly fever Naples virus, mosquito-borne Tahyna virus, Inkoo virus, Batai virus and tick-borne Uukuniemi virus cause aseptic meningo-encephalitis or febrile disease in Europe. Currently, the microarray technology is developing rapidly and there are many efforts to apply it to infectious diseases diagnostics. In order to arrive at an assay system useful for high throughput analysis of samples from aseptic meningo-encephalitis cases the authors developed a combined multiplex ligation-dependent probe amplification and flow-through microarray assay for the detection of European Bunyaviruses. These results show that this combined assay indeed is highly sensitive, and specific for the accurate detection of multiple viruses.     

The epidemic of Q fever in 2018 to 2019 in Zhuhai city of China determined by metagenomic next-generation sequencing

Q fever is a worldwide zoonosis caused by Coxiella burnetii (Cb). From January 2018 to November 2019, plasma samples from 2,382 patients with acute fever of unknown cause at a hospital in Zhuhai city of China were tested using metagenomic next-generation sequencing (mNGS). Of those tested, 138 patients (5.8%) were diagnosed with Q fever based on the presence of Cb genomic DNA detected by mNGS. Among these, 78 cases (56.5%) presented from Nov 2018 to Mar 2019, suggesting an outbreak of Q fever. 55 cases with detailed clinical information that occurred during the outbreak period were used for further analysis. The vast majority of plasma samples from those Cb-mNGS-positive patients were positive in a Cb-specific quantitative polymerase chain reaction (n = 38) and/or indirect immunofluorescence assay (n = 26). Mobile phone tracing data was used to define the area of infection during the outbreak. This suggested the probable infection source was Cb-infected goats and cattle at the only official authorized slaughterhouse in Zhuhai city. Phylogenic analysis based on genomic sequences indicated Cb strains identified in the patients, goat and cattle were formed a single branch, most closely related to the genomic group of Cb dominated by strains isolated from goats. Our study demonstrates Q fever was epidemic in 2018–2019 in Zhuhai city, and this is the first confirmed epidemic of Q fever in a contemporary city in China.     

Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1

Background

Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak.

Results

Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5%) were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19%) dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified.

Conclusion

This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.     

Pestivirus Infections in Norway. Serological Investigations in Cattle,Sheep and Pigs

Serum samples from 1133 dairy cows (187 herds), 3712 ewes (103 flocks) and 1317 adult pigs (877 herds), were tested for neutralizing antibodies against the NADL strain of bovine virus diarrhoea virus. The prevalence rate of seropositive animals was 18.5% in cattle, 4.5% in sheep and 2.2% in pigs, such seroreactors being found in 28 % of the cattle herds and 18 % of the sheep flocks. In all three species the rate showed considerable herd and geographical variation. In cattle the seroreactor rate was similar in herds with normal reproduction and in 62 herds with problems of repeat breeding. Of 31 pig sera containing antibodies against the NADL strain, 27 were also positive in a neutralization test for antibodies against swine fever virus (Baker strain). However, all sera showed a higher titre of antibodies against the bovine strain than against the swine fever virus. It was concluded that the immune response of the pigs had been induced by ruminant pestivirus, and not by swine fever virus.     

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。