2008年甘肃省部分地区虫媒病毒调查

目的调查甘肃省部分地区虫媒病毒分布状况,为虫媒病毒病防治提供依据。方法2008年7-8月在甘肃省嘉峪关、张掖、酒泉、白银和平凉市采集蚊虫标本,用细胞培养法分离病毒,用血清学和分子生物学方法对病毒分离物进行鉴定;利用生物信息学技术对新分离病毒的序列进行同源性和系统发生分析。结果采集到3属280批共13839只蚊虫标本,从中分离到6株病毒,血清学和分子生物学鉴定结果显示6株病毒(GSBY0801、GSBY0804、GSBY0810、GSBY0816、GSBY0827、GSBY0861)均为基因Ⅰ型乙脑病毒。新分离株与减毒活疫苗株SA14-14-2相比,E基因核苷酸同源性为87.5%～87.9%,氨基酸同源性为96.8%～97.2%。结论甘肃省东部地区存在基因Ⅰ型乙脑病毒的流行,流行病毒株与中国四川省乙脑病毒分离株进化关系较近。     

云南新分离四株流行性乙型脑炎病毒全基因组序列特征研究

目的 对云南省2009年和2010年分离的4株流行性乙型脑炎(乙脑)病毒进行全基因组序列测定和分析,阐明近期乙脑病毒流行株的分子生物学特征及基因型。 方法 通过反转录-聚合酶链反应和核苷酸序列测定方法获得病毒全基因组序列,采用Clustal X、DNAstar和Mega等生物学软件进行核苷酸和推导氨基酸序列分析及系统进化分析。 结果 2009年和2010年在云南省中部和西部地区三带喙库蚊(Culex tritaeriorhynchus)中分离到4株乙脑病毒,其中YN09M57株基因组全长10 967 bp,DH10M865、DH10M978和DHL10M62株基因组全长均为10 965 bp,均编码3432个氨基酸。这4株病毒核苷酸和氨基酸同源性分别为98.3%～99.9%和99.5%～99.9%,与来自GenBank的7株基因Ⅰ型乙脑病毒核苷酸和氨基酸同源性分别为98.3%～99.9%和99.7%～99.9%;与基因Ⅱ、Ⅲ、Ⅳ和Ⅴ型参考株核苷酸和氨基酸同源性分别在78.7%～89.7%和91.4%～98.4%;与乙脑减毒活疫苗SA14-14-2株在E蛋白编码区有15个氨基酸位点差异,均位于抗原关键位点之外。基于E基因、全基因组系统进化分析显示云南4株乙脑病毒均为基因Ⅰ型,并形成2个进化分支,分别与相邻省份和东南亚流行株进化关系较近。 结论 云南新分离4株乙脑病毒属基因Ⅰ型,虽然它们之间及其与该型参考株核苷酸和氨基酸位点存在某些差异,但决定病毒毒力和抗原性的关键位点未见明显变化。本研究提示这些乙脑病毒流行株具有稳定的遗传特性和地域特征。     

2009年江西省新分离流行性乙型脑炎病毒株全基因组分子生物学特性研究

目的对2009年分离自江西省蚊虫标本的流行性乙型脑炎(乙脑)病毒JX0939株进行全基因组序列测定,分析其基因组特征。方法使用针对乙脑病毒全基因组测序引物进行PCR扩增基因组片段,PCR产物直接测序,拼接后获得全基因组序列。利用生物信息学软件进行核苷酸序列及氨基酸序列分析和病毒的系统进化分析。结果新分离乙脑病毒JX0939株基因组全长10965个核苷酸,从97位到10392位,共10296个核苷酸编码一个开放阅读框,编码3432个氨基酸。与GenBank中登录的所有59株乙脑病毒全基因组序列比较发现,其核苷酸总体差异率为1%～17%,氨基酸总体差异率为0.1～5%。与目前使用的减毒活疫苗株SA-14-14-2株相比较,只有88%的核苷酸同源性,全基因组共存在1237个核苷酸差异,83个氨基酸差异。提取GenBank登录的及本实验室测定的不同省份的乙脑病毒全基因组序列,进行全基因组序列系统进化分析显示JX0939株属于基因1型乙脑病毒。结论新分离的乙脑病毒JX0939株属于基因1型,与2007年中国分离株SH17M-07进化关系最接近,与疫苗株SA-14-14-2相比关键位点氨基酸未见变异。     

2005年广西媒介蚊虫中流行性乙型脑炎病毒的分离鉴定

目的对广西流行性乙型脑炎(乙脑)流行区的蚊虫进行乙脑病毒分离鉴定。方法2005年6、7月在北流市和靖西县居民区的畜圈内采集蚊虫标本,用C6/36和BHK21细胞分离乙脑病毒,用ClustalX(1.8)、Mega3.1等软件进行分离株E基因序列比对和系统进化分析。结果两地共采集三带喙库蚊771只、骚扰阿蚊425只,从三带喙库蚊中分离到3株基因1型乙脑病毒,3株病毒之间E基因片段核苷酸和氨基酸的最大同源性为99.3%和99.8%,与越南毒株之间核苷酸和氨基酸的最大同源性为98.4%和99.6%。结论从2005年采集自广西的蚊虫中分离到3株基因1型乙脑病毒,这是首次报道在广西分离到基因1型乙脑病毒。     

云南省保山市蚊虫中检测到基因Ⅰ型流行性乙型脑炎病毒

目的 调查云南省保山市蚊虫及其自然感染虫媒病毒状况，为蚊媒病毒病防控提供参考依据。方法 2015年9月采用诱蚊灯法在保山市隆阳区农村人房和畜圈采集蚊虫标本，反转录聚合酶链反应（RT-PCR）用于检测蚊虫标本中黄病毒属、甲病毒属、布尼亚病毒属和流行性乙型脑炎病毒（乙脑病毒）等相关病毒核酸，阳性者进行C/prM基因序列测定、同源性和系统进化分析。结果 共捕获蚊虫3种8 202只，三带喙库蚊（Culex tritaeniorhynchus）、中华按蚊（Anopheles sinensis）和骚扰阿蚊（Armigeres subalbatus）构成比依次为88.50%（7 259只）、11.43%（937只）和0.07%（6只）。蚊虫标本分30批进行RT-PCR检测，14批三带喙库蚊中检测到乙脑病毒核酸，并获得14株乙脑病毒C/prM基因核苷酸序列。同源性和进化分析表明，14株乙脑病毒与基因Ⅰ型乙脑病毒同在一个进化分支，并与2007年保山市腾冲县和2010年德宏傣族景颇族自治州（芒市和瑞丽市）的基因Ⅰ型乙脑病毒具有较近的进化关系。结论 三带喙库蚊为保山市的优势蚊种和乙脑病毒主要传播媒介，当地存在基因Ⅰ型乙脑病毒流行，此为近10年保山市首次检测到基因Ⅰ型乙脑病毒。     

广西北流市分离到基因Ⅰ型流行性乙型脑炎病毒

目的从广西流行性乙型脑炎(乙脑)监测点之一的北流市分离乙脑病毒并研究其基因分型。方法2006年在广西北流市采集蚊虫标本,在C6/36和BHK-21细胞上进行病毒分离,对病毒分离物进行多种虫媒病毒抗体的酶联免疫吸附试验,获得的乙脑病毒进行E基因扩增、测序和基因分型。结果在北流市采集骚扰阿蚊2588只和三带喙库蚊230只,分61批进行处理,获得3个病毒分离物,酶联免疫吸附试验与乙脑抗体反应,E基因测序后基因分型显示为基因Ⅰ型乙脑病毒。结论从广西蚊虫标本分离到基因Ⅰ型乙脑病毒。     

浙江省台州市两株狂犬病病毒流行株的基因序列分析

目的分析浙江省台州市2株狂犬病病毒核蛋白及糖蛋白的基因序列,了解狂犬病病毒野毒株与人用及兽用狂犬病疫苗株间的差异。方法以免疫荧光法检测2008年采自台州市的144只犬脑标本,以RT-(nested)PCR法扩增病毒核蛋白及糖蛋白全基因,克隆测序后以生物信息学软件进行遗传特征分析。结果检测到2株狂犬病病毒阳性样品,序列分析表明两样品所携病毒均为基因1型狂犬病病毒,所携病毒的N基因核苷酸及氨基酸同源性均为100%,G基因核苷酸和氨基酸同源性分别为99.8%和99.4%。所携病毒与CTN疫苗株核苷酸同源性较高,N基因与G基因分别为88.8%和85.9%~86.1%,两样品所携病毒与Hep-Flury疫苗株的核蛋白氨基酸同源性最高,为97.6%,CTN次之,为97.1%,与CTN糖蛋白氨基酸同源性较高,为92.3%~92.5%。与诸基因1型狂犬病病毒参考株相比,两样品所携病毒与浙江温州Wz0(H)、衢州株ZJ-QZ及印度尼西亚株病毒同源性最高。系统发育分析表明XY20及JJ22与浙江温州Wz0(H)、衢州株ZJ-QZ以及印度尼西亚株、疫苗株CTN,以及泰国与马来西亚株进化关系最近,而与疫苗株aG、PV、ERA,及标准攻击毒CVS株等进化关系较远。结论两份阳性犬脑所携狂犬病病毒是基因1型狂犬病病毒,但无论是N基因还是G基因的核苷酸序列,以及推导出来的氨基酸序列与已知的1型狂犬病病毒株及疫苗株存在差异。     

2006年山西省南部地区虫媒病毒分离鉴定

目的了解山西省南部地区虫媒病毒的种类及分布特征。方法使用诱蚊灯捕蚊,通过组织培养法分离病毒,并对病毒分离物进行血清学和分子生物学鉴定。结果2009年9月在山西省运城市、临汾市和阳泉市的3个标本采集点采集到3属4种共3436只蚊虫标本,从中分离到5株病毒分离物,鉴定结果显示有3株(SX0621、SX0633、SX0635)为淡色库蚊浓核病毒,其余2株分离物有待进一步鉴定。对CppDNV非结构蛋白NS1和NS2的部分核苷酸序列分析显示,3株山西省新分离株的同源性为100%;与中国其他省市的分离株同源性在99.9%～100.0%之间。系统进化分析提示所有中国分离株均位于一个相对独立的进化分支中,并且与AaeDNV的进化关系较近。结论在山西省首次分离到淡色库蚊浓核病毒,与中国其他地区的分离株同源性较高。     

2013年北京市首例输入性D_9基因型麻疹毒株的分离和鉴定

目的分离并鉴定北京市输入性D9基因型麻疹毒株。方法使用Vero/SLAM细胞,对可疑麻疹输入性病例的咽拭子和尿液标本进行麻疹毒株的分离培养。用反转录-聚合酶链反应扩增麻疹病毒核蛋白(N)基因羧基末端676个核苷酸片段,对扩增产物进行核苷酸序列测定和分析,并以羧基末端450个核苷酸片段构建基因亲缘关系树,进行遗传距离及核苷酸同源性分析。结果该病毒分离株BJCY13026-2和世界卫生组织D9基因型代表株Victoria.AUS(维多利亚.澳大利亚)12.99在基因亲缘性关系树上同属一个分支,核苷酸同源性为95.8%,氨基酸同源性为96%;和其他23个基因型代表株的核苷酸和氨基酸同源性分别在88.1%~95.6%和90.7%~96.7%。和中国大陆目前所使用的麻疹疫苗株沪191相比对,其核苷酸和氨基酸同源性分别为91.1%和90.0%;和中国目前流行的麻疹病毒绝对优势本土基因型H1a基因型代表株相比对,其核苷酸和氨基酸同源性分别为89.4%和91.3%。结论该输入性病例的病毒分离株为麻疹病毒D9基因型。     

1990～2004年上海市甲型流感病毒基因特性的研究

目的研究1990～2004年上海市甲型流感病毒流行株血凝素(HA)基因的特性,探讨流感病毒基因的变异与流感流行的关系。方法采用鸡胚和MDCK细胞两种方法分离甲型流感病毒。提取病毒RNA进行逆转录-聚合酶链反应(RT-PCR)扩增产物纯化,核苷酸序列测定,并用MegAlign软件进行基因种系发生树分析。结果2000年以来上海市流行的甲3亚型与A/Sydney/5/1997(H3N2),A/Wuhan/359/1995(H3N2)相比,HA1区的核苷酸同源性为95.7%～98.6%和96.8%～98.6%。与90年代流行甲3亚型同源性为88.4%～92.8%,氨基酸的替换发生在抗原决定簇A、B、E、D区和受体结合位点(RBS)的左臂。甲1亚型与A/HongKong/1131/1998(H1N1)、A/Shanghai/7/1999(H1N1)相比,核苷酸同源性为97.8%～99.3%,与90年代流行的甲1亚型同源性为96.8%～97.8%。基因种系发生树表明近几年甲型流感病毒与90年代流行株存在基因特性不同的分支。结论2000年以来上海市H1N1亚型的抗原性未发生明显的变异,H3N2亚型流感病毒的基因发生变异使抗原性发生漂移,是近年引起局部地流感暴发的主要因素。     

2009年辽宁省虫媒病毒分离鉴定

目的 调查辽宁省部分地区虫媒病毒的种类.方法 2009年8月,在辽宁省鞍山市、朝阳市、葫芦岛市和锦州北镇市采集蚊虫标本.蚊虫经分组研磨后接种C6/36和BHK-21细胞,连续3代观察细胞病变情况;对细胞病变阳性的分离物,进行抗原性检测及分子生物学鉴定.结果 在辽宁省上述4个城市共采集蚊虫3600只,主要为中华按蚊(47...     

Studies on Japanese encephalitis virus infection of reptiles. II. Role of lizards on hibernation of Japanese encephalitis virus

A series of experiments on the role of lizards as overwintering hosts of Japanese encephalitis virus (JEV) was carried out. Two species of lizards, T. tachydromoides and E. latiscutatus, 2 species of mosquitoes, Cx. p. fatigans and Cx. p. pallens, and 2 strains of JEV, JaGAr#01 and JaGAr 19461, were used in this study. Firstly transmission of JEV from infected mosquitoes to uninfected lizards and from infected lizards to normal mice by the bite of mosquitoes was demonstrated successfully. Cx. pipiens group mosquitoes were found to feed readily on lizards as compared to Cx. tritaeniorhynchus, the primary vector of JEV in Japan. Secondly simulated hibernation of JEV in lizards was carried out under indoor and outdoor conditions. In the outdoor hibernation, lizards were injected with JEV on October 14, 1968, entered in hibernation on October 19 and were recovered from hibernation on April 10, 1969. Viremias were demonstrated in the lizards for a few weeks in late April. Thirdly JEV isolation and HI antibody detection were attempted from blood samples of field-caught reptiles, 7 species of snakes and 3 species of lizards and among amphibians, 2 species of frogs. HI antibody against JEV was found at a rate of 14.3% from E. latiscutatus and 4.0% from T. tachydromoides, though JEV was not isolated from all the blood samples of these cold-blooded animals. The roles of lizards as overwintering hosts of JEV were discussed.     

Studies on Japanese encephalitis virus infection of reptiles. I. Experimental infection of snakes and lizards

Experimental infection of four species of snakes, Rhabdophis tigrinus tigrinus, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon halys, and five species of lizards, Takydromus tachydromoides, Eumeces latiscutatus, Eumeces barbouri, Eumeces marginatus oshimensis and Gekko japonicus, with Japanese encephalitis virus (JEV) was carried out. Evidence of JEV multiplication in snakes was not obtained at least under the conditions used in the present study. All lizards except G. japonicus were infected with JEV by ip injection of virus suspension. The minimum infectious dose for a lizard was around 10(3) MLD50/0.05 ml, and this dose was considered to be proportional to the virus dose which is injected into a host by a vector mosquito at a single bite. Temperature dependence of JE virus growth in the lizards was demonstrated. JEV multiplied slower at 20 degrees C than at 26 degrees C, though the peak titers of viremia were equivalent in both groups of lizards kept at 20 degrees C and 26 degrees C. E. latiscutatus developed viremia with ip injection of a partially attenuated strain, Nakayama NIH which could not infect adult mice by peripheral inoculation. T. tachydromoides and E. latiscutatus were also infected by oral feeding of JEV infected mosquitoes. E. latiscutatus was infected by oral feeding of only one infected mosquito.     

Analysis of cross-reactivity between flaviviruses with sera of patients with Japanese encephalitis showed the importance of neutralization tests for the diagnosis of Japanese encephalitis

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1–4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.     

Analysis of cross-reactivity among flaviviruses using sera of patients with dengue showed the importance of neutralization tests with paired serum samples for the correct interpretations of serological test results for dengue

Dengue is a febrile illness caused by the dengue virus (DENV) that belongs to the genus Flavivirus in the family Flaviviridae. Cross-reactivity between flaviviruses poses a challenge while interpreting serological test results. In the present study, the cross-reactivity of sera of the patients with dengue, who traveled from Japan to DENV-endemic countries, was analyzed by using an enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT). Sixteen serum samples were collected from patients with dengue and were tested for: i) IgM antibodies against Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) using IgM ELISA, ii) IgG antibody against TBEV using IgG ELISA, and iii) neutralizing antibody against ZIKV, WNV, TBEV, and JEV. Among the 16 samples tested using ELISA, seven samples were IgM-positive for at least one of the other flaviviruses, and nine samples were IgG-positive for TBEV. Neutralizing antibody titers (NATs) against ZIKV, WNV, and TBEV were one-fourth or lower than those against the causative DENV in all samples. The NATs against JEV were one-fourth or lower than those against the causative DENV in six convalescent-phase serum sample among the seven convalescent-phase serum samples. The NAT against DENV of the residual one convalescent-phase serum was similar to that against JEV and that against JEV of its relevant acute-phase serum sample. These results showed that NTs with paired serum samples are important to correctly interpret the serological test results for DENV.