甘肃省天水及陇南部分地区虫媒病毒调查

目的对甘肃省天水陇南部分地区的吸血蚊虫进行病毒分离与鉴定。方法2006年8月在当地采集蚊虫标本,分离病毒和对病毒分离物进行血清学和分子生物学鉴定,以软件进行病毒的核苷酸序列比对和系统发生分析。结果分离到19株病毒,鉴定结果显示分离株GS10-2为盖塔病毒,GS42-2为版纳病毒,其余分离株为一种基因组约8 000核苷酸的未知RNA病毒。盖塔病毒分离株3’UTR中具有与已往中国分离株相同的缺失序列和3个特异核苷酸位点。版纳病毒分离株基因组第12片段的进化关系同其它中国分离株有明显差异,位于一条独立的进化枝中。结论在该地区分离到1株盖塔病毒、1株版纳病毒和17株未知RNA病毒;盖塔病毒与以往我国分离株的遗传关系密切,版纳病毒与我国其它地区分离株有明显差异,在进化上相对独立。     

福建省肠道病毒71型分离株（2010FJLY008）全基因组核苷酸序列分析

目的了解肠道病毒71型在福建省的遗传背景,追踪EV71流行过程中可能发生的变异。方法对2010年福建1例手足口病患儿标本中分离到的1株肠道病毒71型分离株（2010FJLY008）进行全基因组核苷酸序列测定,并对基因组序列进行同源性比较及遗传进化分析。结果2010FJLY008株全长7 403bp,核苷酸及编码氨基酸序列同源性比较,均与08年阜阳流行株Fuyang17.08 2同源性最高,整个基因组核苷酸同源性为98.2%,氨基酸同源性为99.5%;全长基因组核苷酸序列遗传进化分析及VP1区基因遗传进化分析,与Fuyang17.08 2株进化关系较为接近,同属于C4a基因亚型。结论2010年福建省EV71型病毒流行株（2010FJLY008）在病毒基因组结构、基因组核苷酸同源性比较、编码氨基酸同源性比较、全长基因组核苷酸序列遗传进化分析及VP1区基因遗传进化分析等方面,均与2008年阜阳流行株（Fuyang17.08 2）关系密切,未产生明显的抗原漂移及变异。     

首次从云南蚊虫分离到辛德毕斯病毒及其鉴定

目的了解云南省虫媒病毒的存在和流行情况。方法2005年8月,在云南省勐海县采集蚊虫,蚊虫标本经研磨后,上清液接种BHK21细胞以分离病毒,用间接免疫荧光试验和RT-PCR等方法进行鉴定,并对病毒的分子生物学特征进行分析。结果采集到蚊虫标本9400只,其中分离到一株对BHK-21细胞能产生明显细胞病变的病毒(MX10)。该病毒经间接免疫荧光试验提示为甲病毒。用甲病毒属特异引物和Sindbis病毒E1基因特异引物对MX10病毒的RT-PCR扩增为阳性,经核酸序列测定分析证实该序列与Sindbis病毒泰国分离株(AF492770)同源性最高,为90.0%;与1987年分离自云南发热患者的YN87448株和1991年新疆按蚊分离株XJ-160的同源性分别为73.1%和72.0%。结论本次从勐海县蚊虫分离到的MX10病毒株为Sindbis病毒,并可能是Sindbis病毒的新亚型或新株系。     

重庆市库蚊标本中宜昌病毒的分离鉴定

目的 对2017年采集自重庆地区蚊虫标本携带的病毒进行分离鉴定。方法 采用组织细胞培养法进行病毒分离,应用高通量测序技术获得病毒分离物的全基因组序列,采用系统发育法进行种类及分子特征分析。结果 库蚊标本来源的病毒阳性分离物 CQFD2017能引起白纹伊蚊细胞C6/36病变,但不能引起金黄地鼠肾细胞BHK-21、非洲绿猴肾细胞Vero病变。 CQFD2017全基因组序列长度为20 893 bp, 包含6个开放阅读框。系统进化分析发现,该毒株位于Nidovirales病毒目Mesoniviridae病毒科的Yichang病毒所在的进化分支,与Yichang病毒(NC_040534)的核苷酸一致性为98.6%,ORF1a和ORF1b区氨基酸一致性分别为99.06%和99.15%,因此将该毒株命名为Yichang病毒CQFD2017株。结论 2017年重庆市库蚊标本分离的CQFD2017株为Yichang病毒。     

广西中越边境口岸地区1株Nam Dinh病毒的分离及鉴定北大核心CSCD

目的分离鉴定广西中越边境口岸地区来源的1株NDiV,并对其进行同源性比较和进化分析。方法将蚊虫标本研磨液接种C6/36和BHK21细胞,提取阳性分离物的病毒RNA,合成cDNA后进行二代测序,获得毒株的全基因组序列,利用Editseq软件识别该序列的ORF和非编码区,采用Bioedit 7.0软件进行同源性比较,采用MegaⅩ软件分别构建全基因组、RDRP和S基因的系统进化树。结果接种1批次三带喙库蚊标本的C6/36细胞出现明显的病变,病毒命名为Ad2019035sd,二代测序显示该毒株的NDiV覆盖率为99.80%;比较Ad2019035sd与Mesoniviridae科参考株全基因组核苷酸同源性在57.9%~98.9%之间,主要蛋白的编码基因核苷酸和氨基酸同源性分别在56.7%~100%和40.0%~99.5%之间,其中与深圳毒株SZ11706Z的同源性最高。全基因组、RDRP和S基因系统进化树均显示毒株Ad2019035sd与两株NDiV毒株在同一进化分支。结论毒株Ad2019035sd为NDiV,与深圳毒株SZ11706Z高度同源,为广西地区NDiV感染情况调查及相关研究提供了基础资料。     

2017年南京市2株肠道病毒71型分离株全基因组序列测定分析

目的 对2017年南京市2株肠道病毒71型分离株进行全基因组序列测定,分析其进化及遗传变异特征,为手足口病疫情的监控与防治提供依据。方法 通过对临床检测EV71阳性的标本进行病毒分离,设计8对特异性引物对病毒全基因组进行PCR分段扩增,经序列测定及拼接获得EV71基因组序列,将其与其他代表毒株序列分别进行核苷酸和氨基酸同源性比对,并进行遗传进化分析。结果 基因组核苷酸同源性分析显示,2株分离株的同源性为94.0%。以EV71 NJ2017iso2作为参照,与2008年安徽流行株Fuyang 17.08-02的同源性最高(96.9%),而与原型株BrCr的同源性较低(79.8%)。同时基于VP1基因和全基因组序列构建的进化树均可以看出,2株分离株与C4亚型代表株聚为一簇,与国内各地既往的C4流行毒株相比,未发生大的变异。结论 2株EV71分离株均属于C4a型,虽病毒核苷酸序列之间差异显著,但大多为无明显的突变,其相应的氨基酸序列的遗传变化相对稳定。     

柯萨奇病毒A组4型分离株全基因序列分析

目的分析1株柯萨奇病毒A组4型(coxsackievirus A4,CV-A4)毒株的全基因组序列和系统进化特征。方法使用细胞培养法对肠道病毒阳性标本中的病毒进行分离。提取病毒RNA,通过RT-PCR法分段扩增全基因组,经测序、比对和组装后获得CV-A4分离株的全基因组序列。利用MEGA7.0软件分别基于全长VP1序列和全基因组序列构建系统发育树;使用Geneious Prime 2020.1.2软件对全基因组及编码区各区段的核苷酸和氨基酸序列同源性进行分析,并对编码区氨基酸变异情况进行比较分析。结果从人类横纹肌肉瘤(human rhabdomyosarcoma, RD)细胞上分离到1株CV-A4分离株,命名为R09220/YN/CHN/2009。基于全长VP1序列的系统进化分析显示,该分离株属于C2基因亚型。在全基因组核苷酸序列上与其同源性最高的是CV-A4毒株CVA4/SZ/CHN/09(核苷酸为95.4%,氨基酸为98.7%)。与C2基因亚型内16株CV-A4代表株的全序列核苷酸相似性为88.4%～95.4%,氨基酸序列相似性为97.5%～98.7%。通过比较C2基因亚型内的23株CV-A4分离株的全基因组氨基酸序列,R09220/YN/CHN/2009共存在23个氨基酸变异位点,其中10个为其独有的变异位点。结论 R09220/YN/CHN/2009为肠道病毒CV-A4,属于C2基因亚型,该亚型在中国大陆流行的CV-A4病毒中占据优势地位。     

江西省汉坦病毒分离及其S和M基因特征分析

目的 对2011年江西省采集的鼠肺标本进行汉坦病毒的分离鉴定,了解分离病毒株的基因特征。方法 采用Vero-E6细胞对阳性鼠肺标本进行病毒分离,采用直接免疫荧光法和RT-PCR方法对毒株进行鉴定。扩增分离株的S、M片段基因进行克隆测序。运用DNAstar软件包和MEGA3.1软件对序列进行分析。结果 从15份标本分离到2株来自褐家鼠的汉城型汉坦病毒。对其中1株病毒的S、M片段进行了克隆和序列测定,其中S片段含1 772个核苷酸,编码429个氨基酸;M片段含3 651个核苷酸,编码1 133个氨基酸。经核苷酸和氨基酸同源性分析,新分离株与国内外汉城型病毒核苷酸同源性94.8 %~98.0%,氨基酸同源性98.2%~99.6%。与汉城型标准株80-39株相比,S片段仅1个氨基酸位点发生变异;M片段有9个氨基酸位点发生变异,糖基化位点的数目和位置没有发生变化。结论 从江西省褐家鼠分离到两株汉城型病毒,病毒基因变异较小,型别相对稳定。     

江西省汉坦病毒分离及其S和M基因特征分析北大核心CSCD

目的对2011年江西省采集的鼠肺标本进行汉坦病毒的分离鉴定,了解分离病毒株的基因特征。方法采用Vero-E6细胞对阳性鼠肺标本进行病毒分离,采用直接免疫荧光法和RT-PCR方法对毒株进行鉴定。扩增分离株的S、M片段基因进行克隆测序。运用DNAstar软件包和MEGA3.1软件对序列进行分析。结果从15份标本分离到2株来自褐家鼠的汉城型汉坦病毒。对其中1株病毒的S、M片段进行了克隆和序列测定,其中S片段含1 772个核苷酸,编码429个氨基酸;M片段含3 651个核苷酸,编码1 133个氨基酸。经核苷酸和氨基酸同源性分析,新分离株与国内外汉城型病毒核苷酸同源性94.8%~98.0%,氨基酸同源性98.2%~99.6%。与汉城型标准株80-39株相比,S片段仅1个氨基酸位点发生变异;M片段有9个氨基酸位点发生变异,糖基化位点的数目和位置没有发生变化。结论从江西省褐家鼠分离到两株汉城型病毒,病毒基因变异较小,型别相对稳定。     

云南登革4型病毒的鉴定及NS1和NS2a基因序列分析

目的对1989年8月分离自云南省盈江县蚊虫的2株登革病毒进行生物学及分子特征的鉴定,明确这两株病毒的血清型及基因型。方法蚊虫用BHK21细胞和乳鼠法分离病毒,用间接免疫荧光试验、RT-PCR等方法进行鉴定,并对这两株病毒的分子生物学特征进行分析。结果从白纹伊蚊和达勒姆阿蚊中各分离到一株病毒,分别命名为M110和M113,这两株病毒对BHK21细胞能产生明显的细胞病变,脑内接种乳鼠4d左右导致乳鼠死亡。该病毒经血凝、血凝抑制和间接免疫荧光试验提示为黄病毒。分别用黄病毒属特异引物、登革4型病毒NS1和NS2a基因片段特异引物进行RT-PCR扩增、序列测定,证实为登革4型病毒。NS1和NS2a基因序列片段分析表明,M110和M113的NS1核苷酸序列与登革4型病毒基因Ⅰ型的H241株(Y19176)同源性最高,分别为99%、98%;NS2a基因片段与H241的核苷酸序列的同源性分别为96.5%、97.1%。结论从盈江县蚊虫分离到的M110和M113病毒为登革4型病毒基因Ⅰ型,表明云南省西部边境地区在20世纪80年代发生过登革4型病毒的流行。     

云南楚雄2014年蚊媒及其病毒感染调查

目的了解云南楚雄元谋、武定和双柏3县蚊虫及其主要虫媒病毒种类,为制定出有效的虫媒传染病防控对策提供依据。方法2014年6-7月采用诱蚊灯在调查点通宵捕捉蚊虫,对现场采集蚊虫进行形态分类鉴定;采用C6/36细胞培养法和RT-PCR扩增目的基因片段对蚊虫体内病毒进行分离、鉴定。结果共捕获4属15种12 384只蚊虫,三带喙库蚊、中华按蚊、致倦库蚊和微小按蚊分别占捕获蚊虫总数的59.95%、19.31%、10.90%和 4.13%;C6/36细胞培养处理116组蚊虫,其中29组发生细胞病变,经基因鉴定为版纳病毒(BAV)、Totivirus 病毒(TOV)、乙型脑炎病毒(JEV)和Nam Dinh病毒(NDiV)4种,其中,三带喙库蚊的JEV、BAV、TOV和NDiV基因扩增批阳性率分别为1.87%、11.32%、3.77%和9.43%;希氏库蚊的NDiV基因扩增批阳性率为25.00%;致倦库蚊和中华按蚊的BAV、NDiV批阳性率分别为20.00%、33.34% 和5.00%、7.50%。结论楚雄地区蚊虫种类多,且以三带喙库蚊和中华按蚊为优势蚊种,从两种蚊虫标本中能分离到JEV、TOV、BAV和NDiV病毒,表明调查地区发生乙脑及其它虫媒病毒性疾病流行的危险较大。     

Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia.

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.     

ZIKA病毒及其蚊虫传播媒介

ZIKA病毒首次于1947年在非洲恒河猴标本分离到,此后于1948年在同一地区采集的非洲伊蚊(Aedes africnus)标本中分离到ZIKA病毒,确定为蚊传病毒。自1948年以来全世界已经在自然界采集的20余种蚊虫(2种按蚊,15种伊蚊,3种库蚊)中分离到ZIKA病毒,且在这些蚊虫中获得ZIKA病毒阳性分离物60余次。与此同时,已经对23种蚊虫开展过人工感染ZIKA病毒的蚊传试验研究,并证实8种(7种伊蚊,1种库蚊)为ZIKA病毒传播媒介。可见ZIKA病毒可以由多种蚊虫携带和传播。目前我国已经在自然界采集的致倦库蚊和骚扰阿蚊中分离到ZIKA病毒,但尚未有本地感染病例报告。因此了解国际上有关蚊虫与ZIKA病毒的研究进展对于我国相关研究工作和ZIKA病毒病预防控制具有重要借鉴意义。     

Genomic and biologic analyses of snowshoe hare virus field and laboratory strains.

Low-passage field strains of snowshoe hare (SSH) virus (Bunyaviridae), the prototype SSH virus (originally isolated in Montana), and La Crosse (LAC) virus were compared serologically by plaque-reduction neutralization (PRNT) and molecularly by oligonucleotide fingerprinting (ONF). The PRNT and ONF results confirmed the identity of the field strains, although some differences in the fingerprints were observed. We have examined the RNA genome variability in the two field and three laboratory strains of SSH virus, using direct sequence analysis of selected RNase T1 oligonucleotides. Few changes were observed among three Montana prototype-derived laboratory isolates, although they have different passage histories. In contrast, the field isolates differed greatly from the laboratory strains. In addition, we have located several of the larger T1 oligonucleotides within the known sequence of the small and large RNA genome segments. We then compared the viruses for their ability to replicate in and be transmitted by Aedes triseriatus mosquitoes. The oral infection rates for LAC, the field isolates, and the SSH prototype, as determined by immunofluorescent examination of midgut tissues, were 100%, 82%, and 47%, respectively. All viruses were also transmissible from mosquitoes to mice.     

Virus-vector-host relationships of Aedes stimulans and Jamestown Canyon virus in a northern Indiana enzootic focus

Collections of hematophagous Diptera at the Kingsbury State Fish and Wildlife Area in northern Indiana between 1982 and 1984 yielded 118,972 mosquitoes from which 5 isolates of Jamestown Canyon virus and 3 isolates of trivittatus virus were obtained. All Jamestown Canyon isolates were from Aedes stimulans, including 1 from a pool of newly emerged males and 2 from pools of newly emerged females. These 3 isolates suggest that Jamestown Canyon virus is transovarially transmitted by Ae. stimulans. All isolates of trivittatus virus came from pools of Ae. trivittatus. No isolates were obtained from greater than 4,000 tabanids collected along with the mosquitoes those years. Transmission trials with field-collected newly emerged adult female Ae. stimulans demonstrated a mean midgut infection rate of 44%, a disseminated infection rate of 16%, and an oral transmission rate of 12% to suckling mice. Precipitin tests of field-collected bloodfed female mosquitoes indicated that white-tailed deer were the preferred host for numerous mosquito species including Ae. stimulans. The results of this study suggest that Ae. stimulans is a primary vector of Jamestown Canyon virus and that transovarial transmission is the probable overwintering mechanism for this California group virus.     

Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes.

Between July and October 1996, a West Nile (WN) fever epidemic occurred in the southern plain and Danube Valley of Romania and in the capital city of Bucharest, resulting in hundreds of neurologic cases and 17 fatalities. In early October 1996, entomologic and avian investigations of the epidemic were conducted in the city of Bucharest and nearby rural areas. Thirty (41%) of 73 domestic fowl sampled had neutralizing antibody to WN virus, including 5 of 13 ducks (38%), 1 of 1 goose, 19 of 52 chickens (37%), 1 of 1 peahen, and 4 of 6 turkeys (67%). Seroprevalence in domestic fowl (27%, or 7 of 26) from the urban Bucharest site was not significantly different (P = 0.08, by Fisher's exact test) than rates at three rural sites (50%, or 23 of 46). Serum collected from one of 12 Passeriformes, an Erithacus rubecula, was positive for neutralizing antibody to WN virus. A total of 5,577 mosquitoes representing seven taxa were collected. Culex pipiens pipiens accounted for 96% of the mosquitoes collected. A single virus isolate, RO97-50, was obtained from a pool of 30 Cx. p. pipiens females aspirated from the walls and ceiling of a blockhouse located near the center of Bucharest, resulting in a minimum infection rate of 0.19 per 1,000. Antisera prepared against RO97-50 failed to distinguish among RO97-50, WN virus strain Eg101, and Kunjin (KUN) virus strain MRM16. A 2,323-basepair DNA fragment of the envelope (E) glycoprotein gene from RO97-50 and a Romanian WN virus strain obtained from a human cerebrospinal fluid sample, RO96-1030, were sequenced. Phylogenetic analyses of 23 WN virus strains and one KUN virus strain using the amino acid and nucleotide sequences for a small portion of the E gene suggest the existence of two large lineages of viruses. Bootstrap analysis of the nucleotide alignment indicated strong support (95%) for a lineage composed of WN virus strains from northern Africa, including isolates from Egypt and Algeria, and west, central, and east Africa, all of the European isolates, those from France and Romania, an Israeli isolate, and an isolate of KUN virus from Australia. The nucleotide sequence of RO97-50 was identical to the sequence of a WN virus isolate obtained from Cx. neavei mosquitoes from Senegal and Cx. univittatus mosquitoes from Kenya. The phylogenetic analyses were compatible with the introduction of virus into Romania by birds migrating from sub-Saharan Africa, to northern Africa, and into southern Europe.     

An outbreak of human Semliki Forest virus infections in Central African Republic

Semliki Forest (SF) virus was responsible for an outbreak of febrile illnesses in Bangui, Central African Republic (CAR), during October-December 1987. The virus was isolated at first from mosquitoes, mainly Aedes africanus, collected August-October in a gallery forest 100 km from Bangui. During October-December, 22 isolations of SF virus were made from serum samples collected from patients in Bangui presenting with fever, severe persistent headache, myalgia, arthralgia, and a convalescence marked by asthenia. During the same period, 8 SF virus isolates were obtained from mosquitoes collected in Bangui, mainly from Ae. aegypti. Europeans, particularly soldiers who had recently arrived from France, were affected. Antibodies to alphaviruses had been previously detected in a high proportion of resident human populations in CAR. We conclude that SF virus is a human pathogen.     

Viruses isolated from Aedeomyia squamipennis mosquitoes collected in Panama, Ecuador, and Argentina: establishment of the Gamboa serogroup

Twenty-four virus strains were isolated from Aedeomyia squamipennis mosquitoes collected in Ecuador. One additional strain each was isolated from this species from Panama and ARgentina. All 26 isolates were shown to be related serologically to prototype Gamboa virus, originally isolated from Ad. squamipennis mosquitoes collected in Panama. Antigenic comparisons of eight strains, including prototype Gamboa virus, indicated the existence of four distinct viruses. Neutralization tests with sera from a variety of mammalian and avian species from Argentina provided further evidence that Gamboa serogroup viruses are transmitted between Ad. squamipennis and birds.