上海市2002年病毒性脑炎病因病毒的分离和鉴定

采集上海市 2 0 0 2年 6 9例病毒性脑炎散发病人的脑脊液 (CSF)标本 ,用Hep 2、RD、Vero细胞分离出肠道病毒(EV) 2 1株 ,经用标准血清鉴定为柯萨奇病毒B5(Coxsackie ,Cox B5) 11株 ,Cox B2 1株 ,Cox A161株 ,埃可病毒 (E CHO) 3株 ,非脊髓灰质炎肠道病毒 (NPEV) 5株 ,Cox B5病毒分离率为 16 %。本次调查结果表明 ,Cox B5病毒是上海市 2 0 0 2年病毒性脑炎的主要病原。     

引发脑膜脑炎流行的柯萨奇B5病毒的序列分析

安徽省明光市 2 0 0 1年 6～ 7月发生了一起无菌性脑膜脑炎流行 ,从病人脑脊液和粪便标本中分离到 17株柯萨奇 (Coxsackie ,Cox .)B5病毒 ,血清学实验证明所分离的病毒为此次无菌性脑膜脑炎流行的病因。对其中的 2株病毒 (MG/ 18/ 2 0 0 1和MG/ 39/ 2 0 0 1)测定了VP1区全基因核酸序列并做了比较分析。结果显示 :所测 2株病毒VP1区核苷酸长度均为 849bp ,两毒株之间仅有 1个核苷酸不同。这两株病毒与从 (GenBank)检索到的Cox B5病毒的核苷酸同源性约为 82 %,氨基酸序列亦有改变。从进化树上看 ,这两株病毒同在一个小分支上 ,与它们亲缘关系最近的是从一疑似脊髓灰质炎的儿童分离到的AF114383 -Cox B5病毒 ,而与从手足口病患者分离到的X6 770 6-Cox B5病毒相对较远。表明此次脑膜脑炎的病因病毒与同是侵犯中枢神经系统的Cox B5的原型株 -AF114383 -Cox B5病毒亲缘关系最近 ,但核苷酸已经发生了 >18%的变异 ,可以认为其是Cox B5新亚型病毒。     

肠道病毒ECHO 19型引起无菌性脑炎流行的诊断与遗传性分析

目的明确引起无菌性脑炎流行的病原,了解其遗传特征.方法收集部分病例脑脊液及双份血清标本,同时应用传统的病毒分离及中和鉴定、RT-PCR扩增肠道病毒目的基因片段及序列分析和双份血清中和抗体变化3种方法进行病因学诊断;并进一步针对病原进行基因序列测定和遗传树图构建分析.结果直接从首例及随后送检的18份脑脊液标本中扩增出肠道病毒核酸进行了早期诊断;病毒分离鉴定及双份血清中和抗体检测结果为：用RD和Hep-2两种细胞从30份脑脊液标本中分离出肠道病毒ECHO 19型16株（阳性分离率为53.33%）,检测5例病例双份血清ECHO 19病毒中和抗体水平,其中4例双份血清抗体阳转或呈4倍及以上升高,同时也证实了肠道病毒ECHO 19为此次脑炎流行的病因;其中5株病毒VP1区全基因序列与已发表的其他ECHO 19病毒株进行比对分析,显示：本次分离株间核苷酸序列同源性为98.9%～100.0%,来自于一个共同的祖先,而与已发表其他毒株间亲缘性较远,差异性为13.0%～22.4%,属于一新的变异福建株.结论此次无菌性脑炎流行的病原为一变异的ECHO 19病毒;运用分子生物学技术不仅可以达到早期诊断目的,而且可以从分子水平提示病毒的遗传特性.     

一起暴发性病毒性脑炎的病原学调查

目的 分离鉴定引发2010年河南省一起病毒性脑炎暴发的病原,并进一步分析该病原体分离株外壳蛋白1(VP1)区基因特征。方法 于2010年4月河南省平顶山市鲁山县病毒性脑炎暴发期间,采集8例住院患儿的粪便标本（共8份）,其中3例患儿同时采集脑脊液标本（共3份）,未采集到脑脊液标本的5例患儿中4例采集了血清标本（共4份）。对标本进行病毒分离和肠道病毒(EV)组合血清鉴定,对阳性分离物采用逆转录PCR方法扩增VP1区基因、测定核苷酸序列并进行亲缘进化分析。结果 采集的脑脊液、血清、粪便标本共15份,荧光定量PCR检测结果显示,11份标本EV通用检测阳性,其中2份粪便标本分离到2株病毒毒株,EV组合血清鉴定血清型别均为柯萨奇病毒(Cox)阳性,VP1区全长基因序列均为849 bp,与Cox B5的核苷酸相似性为77.1％～96.9％,氨基酸相似性为95.8％ ～ 100％,遗传发育树分析与D基因型属同一分支。结论 CoxB5是引起本次河南省平顶山市鲁山县病毒性脑炎暴发的病原体,其基因型为D基因型。     

上海市手足口病患者肠道病毒71型的分离

2 0 0 0年 10～ 11月 ,上海市杨浦区某幼儿园发生手足口病流行 ,采集了 2 5例粪便标本分离病毒 ,用RD细胞分离出肠道病毒 (EV) ,经用标准血清鉴定为EV71型 13株 ,埃可病毒 13型 (ECHO13 ) 1株 ,EV71型分离率为 5 2 %。病人血清与EV71型分离株的中和抗体滴度较高 ,多数 >1∶2 5 6 ,而与柯萨奇病毒A组 16型 (Cox .A16)的中和试验滴度多数 <1∶2。 5例双份血清有 1例EV71型抗体呈 4倍升高 ,而Cox .A16型均无 4倍升高 ,表明本次流行由EV71型引起。     

浙江省1996～1999年急性弛缓性麻痹病例中非脊髓灰质炎肠道病毒分析

浙江省1996～1999年急性弛缓性麻痹（AFP）病例粪便标本中非脊髓灰质炎（脊灰）肠道病毒（NPEV）的平均分离率为11.2％。在67株NPEV中,柯萨奇病毒（Coxsackievirus,Cox.）B组21株,占31.3％;埃可病毒（EntricCytopothicHumanOrphanviruses,ECHO）25株,占37.4％;不能确定型别21株,占31.3％。NPEV按月分布显示,Cox.B病毒高峰期在5～7月,在1～3、10、12月未分离到;ECHO病毒全年均有,高峰期在8～11月。Cox.B病毒在Hep－2、RD细胞上平均滴度分别为10     

浙江省2008年病毒性脑膜脑炎病原柯萨奇B组5型病毒VP_1区基因变异分析

目的研究浙江省2008年病毒性脑膜脑炎中分离到的柯萨奇B组5型病毒(Coxsackie Virus B5,CVB5)VP1区的基因特征,并与其他国家的分离株和CVB5原型株进行比较,探讨病毒VP1区的变异与病毒性脑膜脑炎流行的关系。方法用人喉癌上皮细胞和人横纹肌肉瘤细胞对脑脊液(Cerebrospinal Fluid,CSF)和粪便标本进行病毒分离,对分离到的病毒株提取核糖核酸,再用逆转录-聚合酶链反应扩增CVB5VP1区基因片段并进行序列测定,用DNA MAN和Bioedit等软件进行分析处理。结果从浙江省2008年病毒性脑膜脑炎患者CSF和粪便标本中,分离到的5株CVB5,其VP1区的核苷酸长度均为735bp,未发现核苷酸插入与丢失。与浙江(Zhejiang,ZJ)/12/02株氨基酸同源性最高为99.6%～100%,与法国等国外毒株的同源性为97.3%～99.6%,而与CVB5原型株的同源性只有96.3%。不同年份、不同地点分离的CVB5在进化树上显示在同一个分支上。结论2008年引起浙江省病毒性脑膜脑炎的CVB5的VP1区变异较小,引起的病毒性脑膜脑炎在局部地区散发,未发生明显的流行。     

浙江省肠道病毒71型的分离与VP1区域序列分析

目的研究浙江省手足口病患者中肠道病毒（EV）71型分离株的病毒基因特征。方法采集手足口病患者粪便、疱疹液和咽拭子标本，进行病毒分离和逆转录一聚合酶链反应（RT—PCR）特异性扩增进行鉴定，同时选取其中6株EV71分离毒株，对其抗原决定簇部位VP1区进行核苷酸序列测定并参考EV71A、B、C各基因型的参考毒株和以往中国EV71的分离毒株进行同源性分析和构建系统发生树。结果从14份标本中分离出EV9株，经中和试验和RT—PCR特异性扩增，均证实为EV71。其中6株EV71分离毒株与A、B基因型参考毒株的核苷酸和氨基酸同源性分别为82．9％～85．5％和94．9％～98．0％；与C基因型比较，同源性分别为89．2％～94．1％和97．0％～99．0％。而与C基因型的C1、C2、C3、C4亚型代表株的核苷酸同源性分别为91．0％～92．2％、90．2％～90．3％、89．2％～89.5％、96．7％～96．9％，其中与国内以往分离到的C4亚型毒株的核苷酸同源性可达93.8％～97．1％。在系统发生树上，这6株毒株均在C基因型中，与属C4亚型的11株毒株属同一分支。结论浙江省手足口病患者中分离的EV71毒株属C基因型的C4亚型。     

2006-2008年汕头市病毒性脑炎病原监测

目的通过对汕头市病毒性脑炎的病原监测,了解该地区病毒性脑炎的病原谱及发病情况。方法哨点医院对2006-2008年就诊的疑似脑炎病例进行血液及脑脊液标本采集,运送至汕头市疾病预防控制中心,采用ELISA法对脑炎疑似标本进行乙脑病毒(JEV)IgM、单纯疱疹病毒(HSV)IgM、巨细胞病毒(CMV)IgM、科萨奇病毒(COXV)IgM、埃可病毒(ECHO)IgM、腺病毒(ADV)IgM 6种病毒抗体项目检测。结果监测期间共接收148例疑似脑炎病例,采集的标本总共检出病毒抗体阳性标本19份,病毒总阳性率为12.8%。其中JEV阳性2份;HSV阳性6份;CMV抗体阳性1份;ECHO抗体阳性1份;ADV抗体阳性7份;HSV和ADV抗体阳性1份;ECHO和ADV抗体阳性1份。结论汕头市病毒性脑炎病原以ADV和HSV阳性率较高,需加强此类病毒的防控。     

腮腺炎病毒抗体检测在腮腺炎并发脑膜脑炎诊断中的应用

目的　分析腮腺炎特异性IgM抗体检测对腮腺炎流行期间并发的脑膜脑炎的诊断价值。 方法　对某市 2 0 0 3年 4～ 8月间发生的 35 6例腮腺炎及其中并发典型症状的 83例脑膜脑炎患者进行分析 ,其中 2 3例脑膜脑炎患者的血清或脑脊液用ELISA法作特异性IgM抗体检测。结果　 2 73例腮腺炎病人病症典型 ;83例脑膜脑炎者大多数腮腺炎疫苗免疫史不祥 ,有 1 5例伴典型的腮腺炎症状 ,2 3例脑膜脑炎患者标本腮腺炎特异性IgM抗体检测 ,在 1 2份血清中有 6份阳性 ,1 3份脑脊液中有 2份阳性。结论　腮腺炎特异性IgM抗体检测技术对腮腺炎引起脑膜脑炎诊断有参考价值     

Global distribution of rubella virus genotypes

Phylogenetic analysis of a collection of 103 E1 gene sequences from rubella viruses isolated from 17 countries from 1961 to 2000 confirmed the existence of at least two genotypes. Rubella genotype I (RGI) isolates, predominant in Europe, Japan, and the Western Hemisphere, segregated into discrete subgenotypes; international subgenotypes present in the 1960s and 1970s were replaced by geographically restricted subgenotypes after approximately 1980. Recently, active subgenotypes include one in the United States and Latin America, one in China, and a third that apparently originated in Asia and spread to Europe and North America, starting in 1997, indicating the recent emergence of an international subgenotype. A virus that potentially arose as a recombinant between two RGI subgenotypes was discovered. Rubella genotype II (RGII) showed greater genetic diversity than did RGI and may actually consist of multiple genotypes. RGII viruses were limited to Asia and Europe; RGI viruses were also present in most of the countries where RGII viruses were isolated.     

Induction of protective immunity in swine by immunization with live attenuated recombinant pseudorabies virus expressing the capsid precursor encoding regions of foot-and-mouth disease virus

Foot-and-mouth disease (FMD) causes morbidity to livestock and serious economic consequences to its associated industry and therefore it is necessary to develop a safe and efficient vaccine to prevent or control this disease. A recombinant live attenuated virus vaccine, designated PRV-P1, was generated by insertion of an expression cassette containing CMV promoter, FMDV P1 gene and SV 40 poly-A into the gG gene region of a live attenuated pseudorabies virus vaccine strain (TK⁻/gG⁻/LacZ⁺). To determine the induction of protective immunity, 16 FMDV and PRV seronegative white swine were randomly divided into four groups and immunized intramuscularly. The parental virus (TK⁻/gG⁻/LacZ⁺) was injected into three pigs, the recombinant virus PRV-P1 into five pigs and commercial FMD-inactivated vaccine into five pigs, with PBS (negative control) into three pigs. All animals were immunized again 4 weeks later to boost the immune response and challenged with virulent type O FMDV O/ES/2001 strain 4 weeks after the second immunization. Results showed PRV-P1 vaccinated pigs induced high-level neutralizing antibody response to both FMDV and PRV, and strong CTL response against FMD antigen activation. Three of five pigs were completely protected against challenge with FMDV, one pig minimally protected and the other one had increased protection but not complete. However, one pig vaccinated with commercial FMD vaccine developed constant pyrexia. Average levels of antibodies against non-structural 3ABC proteins were significantly lower and efficacy on inhibition of FMDV replication was much increased in swine vaccinated with PRV-P1 than those immunized with commercial FMD vaccine after FMDV challenge. Our results showed that the recombinant PRV-P1 can induce not only humoral and cell-mediated immune responses but also partial protection against FMDV challenge, making it a good candidate for future development of the FMD vaccine.     

海南省2017-2018年虫媒病毒的分离与鉴定

目的了解海南省虫媒病毒种类及分布变化。方法于2017-2018年在海南省野外采集吸血昆虫标本,所有吸血昆虫标本实验室处理后用BHK-21细胞和C6/36细胞进行病毒分离,同时使用RT-PCR平行检测吸血昆虫标本中虫媒病毒基因。结果共采集到4属(库蚊、阿蚊、伊蚊、按蚊)15062只蚊虫和11360只蠓虫。采集的蚊虫中三带喙库蚊居多,占蚊虫采集总数92.88%(13990/15062)。经组织培养细胞共获得4株病毒分离物,其中3株乙型脑炎(乙脑)病毒、1株盖塔病毒,在5批三带喙库蚊标本中检测到乙脑病毒基因阳性。遗传进化分析结果显示这3株乙脑病毒和5批PCR筛检阳性的标本均属于基因1型乙脑病毒。当地乙脑病毒的最低感染率为0.57‰(8/13990)。在5批蠓虫标本中检测阿卡斑病毒基因阳性,当地阿卡斑病毒的最低感染率为0.44‰(5/11360)。基于病毒S基因和M基因序列的系统进化分析显示这5株阿卡斑病毒处于单独进化分支形成独特的地理分布特征。结论继1980年代以来,从海南省的蚊虫标本中再次分离到乙脑病毒和盖塔病毒、蠓虫标本中检测到阿卡斑病毒。应加强乙脑病毒、盖塔病毒和阿卡斑病毒对人畜动物感染状况及疾病负担的监测,以减少对当地公共卫生健康的危害。     

Prevalence of hepatitis virus infections in Albanian refugees

A sample of 393 Albanian refugees, including both children and adults, was tested for serological HAV, HBV, HDV and HCV markers. A high prevalence of infection with both the hepatitis A and B viruses was found, while HDV and HCV infections were uncommon. The overall prevalence of anti-HAV was 96%; it was very high in children 0-10 years, suggesting that HAV infection is largely acquired during childhood and that poor ambiental conditions influence the spreading of this viral infection.One or more serological markers of HBV infection were found in 295 Albanians (75%), confirming the endemic nature of this virus in the Albanian community. The overall prevalence of HBsAg was 19%, and the carrier rate was higher in males than in females. The high HBsAg prevalence among children suggests that HBV infection is usually acquired in early childhood.The serological data obtained in the Albanian sample examined clearly indicate the urgent need for measures to reduce the incidence of HAV and HBV infections and to avoid the further spread of HDV and HCV infections.Finally, the high prevalence of type B hepatitis indicates the necessity of vaccination against HBV for all risk groups and for all children at birth.     

48例静脉药瘾者多重病毒感染临床分析

目的探讨静脉药瘾病毒性肝炎患者多重病毒感染率及转归。方法对48例静脉药瘾病毒性肝炎患者进行乙型肝炎病毒（HBV）、丙型肝炎病毒（HCV）、人免疫缺陷病毒（HIV）血清标志物及核酸测定,并进行临床分析。结果48例静脉药瘾病毒性肝炎患者中,HBV、HCV双重感染率为31.25%,HBV、HIV双重感染率为4.17%,HBV、HCV、HIV三重感染率为62.50%;重型肝炎者占70.83%。重型肝炎患者住院时间为（48.67±19.25）d,与同期住院的非药瘾重型肝炎患者住院时间（65.32±31.49）d相比,明显缩短（U=4.25,P＜0.01）。结论静脉药瘾者病毒重叠感染率高,且重型肝炎患者多,但相对非药瘾重型肝炎患者恢复较快。     

Monitoring the uptake of live avian vaccines by their detection in feathers

Protection against diseases caused by the avian viruses, Marek's disease, Infectious laryngotracheitis, chicken anemia and turkey meningoencephalitis is achieved by live vaccines. The application quality is important to assure proper uptake in commercial flocks. We describe a novel evaluation method for the vaccination process by sequential monitoring the vaccine viruses in feathers. Feather collection is easy, non-invasive and non-lethal for the birds, therefore advantageous for monitoring purposes. To demonstrate the vaccine virus presence, an innovative assay of nested real-time amplification was approached because vaccine viruses presence in vivo is less abundant comparing to virulent wild-type isolates.The Marek's disease virus vaccine virus, Rispens/CVI988, in feathers of commercial flock was detected from 4 to 7 days and for at least 3 months post-vaccination, until the survey stopped. As the drinking water route was newly adopted for Infectious laryngotracheitis vaccination, one or two vaccine doses/bird were administered. The virus uptake was detected in feathers between 2 and 20 days-post-vaccination. With a doubled vaccine dose the positivity bird rate was higher. For the first time the chicken anemia vaccine virus presence in chicken feathers was demonstrated between 14 and 35 days-post-vaccination. No previous studies were available, thus in parallel to feathers the vaccine virus was demonstrated in the livers and spleens. The turkey meningoencephalitis vaccine virus uptake in turkey feather-pulps is even more innovative because this is the first turkey virus amplified from feather-pulps. The vaccine virus presence resemble the kinetics of the other 3 viruses, 3–21 days-post-vaccination. Detecting the specific antibodies following vaccination possessed a lower sensitivity than vaccine virus demonstration in feathers. In summary, the presented assay can be adopted for the quality evaluation of the vaccination process in poultry.     

多重逆转录-聚合酶链反应检测麻疹风疹流行性腮腺炎病毒基因的研究

目的　探索麻疹、风疹、流行性腮腺炎 (腮腺炎 )病毒感染的快速诊断方法。方法　利用多重逆转录 聚合酶链反应 (MRT PCR)方法对麻疹、风疹、腮腺炎病毒的相关基因检测进行研究。结果　直接从麻疹疫苗沪1 91 株 ,风疹疫苗BRDⅡ株 ,腮腺炎疫苗S79株 ,冻干麻疹、腮腺炎、风疹联合减毒活疫苗及临床麻疹、风疹、腮腺炎患者的标本中 ,同时检测出麻疹、风疹、腮腺炎病毒 6 3 5bp、4 1 1bp、5 1 9bp的特异性核酸片段 ,MRT PCR 1次扩增灵敏度均可分别达到 1TCID50 。结论　该方法能特异、敏感地检测临床标本中麻疹、风疹、腮腺炎病毒 ,是快速诊断麻疹、风疹、腮腺炎病毒感染的理想方法     

Recombinant varicella vaccines induce neutralizing antibodies and cellular immune responses to SIV and reduce viral loads in immunized rhesus macaques

The development of an effective AIDS vaccine remains one of the highest priorities in HIV research. The live, attenuated varicella-zoster virus (VZV) Oka vaccine, safe and effective for prevention of chickenpox and zoster, also has potential as a recombinant vaccine against other pathogens, including human immunodeficiency virus (HIV). The simian varicella model, utilizing simian varicella virus (SVV), offers an approach to evaluate recombinant varicella vaccine candidates. Recombinant SVV (rSVV) vaccine viruses expressing simian immunodeficiency virus (SIV) env and gag antigens were constructed. The hypothesis tested was that a live, attenuated rSVV-SIV vaccine will induce immune responses against SIV in the rhesus macaques and provide protection against SIV challenge. The results demonstrated that rSVV-SIV vaccination induced low levels of neutralizing antibodies and cellular immune responses to SIV in immunized rhesus macaques and significantly reduced viral loads following intravenous challenge with pathogenic SIVmac251-CX-1.     

Tickborne arbovirus surveillance in market livestock, Nairobi, Kenya

To identify tickborne viruses circulating in Kenya and the surrounding region, we conducted surveillance at abattoirs in Nairobi, Kenya. Species of ticks collected included Rhipicephalus pulchellus (56%), Amblyomma gemma (14%), R. appendiculatus (8%), A. variegatum (6%), and others. A total of 56 virus isolates were obtained, 26 from A. gemma, 17 from R. pulchellus, 6 from A. variegatum, and 7 from other species. Virus isolates included Dugbe virus (DUGV), an unknown virus related to DUGV, Thogoto, Bhanja, Kadam, Dhori, Barur, and foot-and-mouth disease (FMDV) viruses. This is the first report of Dhori virus isolation in East Africa and the first known isolation of FMDV associated with tick collection. Our results demonstrate the potential for tickborne dissemination of endemic and emergent viruses and the relevance of A. gemma in the maintenance of tickborne viruses in this region.     

Arboviruses: A global public health threat

A conference on «ARBOVIRUSES, A GLOBAL PUBLIC HEALTH THREAT» was organized on June 20–22, 2018 at the Merieux Foundation Conference Center in Veyrier du Lac, France, to review and raise awareness to the global public health threat of epidemic arboviruses, and to advance the discussion on the control and prevention of arboviral diseases. The presentations by scientists and public health officials from Asia, the Americas, Europe and Africa strengthened the notion that arboviral diseases of both humans and domestic animals are progressively becoming dominant public health problems in the world. The repeated occurrence of recent deadly epidemics strongly reinforces the call for action against these viral diseases, and the need for developing effective vaccines, drugs, vector control tools and strong prevention programs.