用温度循环法测定扩增DNA的序列比较汉坦病毒同型间的核酸序列

用温度循环法测定扩增ＤＮＡ的序列比较汉坦病毒同型间的核酸序列作者应用温度循环测序法对汉坦病毒血清型特异性引物扩增所得的ＰＣＲ产物进行序列测定以确定同一血清型几株病毒间序列的相似水平。测序用模板为上游引物和下游引物之间,测得的２８２,２６４和２９１个碱...     

多聚酶链反应技术鉴别肾综合征出血热病毒血清型的初步研究

肾综合征出血热(HFRS)为一组抗原性密切相关的布尼亚科汉坦病毒引起的急性传染病。在我国存在至少两种临床表现、动物宿主及流行特征截然不同的血清型别,即血清Ⅰ型(汉坦型)和血清Ⅱ型(汉城型)。这两型病毒间的血清学定型已有报道。近年来,除啮齿类动物外,从临床病人以及非啮齿类动物体内也分离到了HFRS病毒。同时出现两类型别毒株共存,以及从家鼠体内分离到野鼠型毒株或从野鼠体内分离到家鼠型毒株的复杂情形。为此,准确检定并鉴别不同来源毒株型别,将为深入了解其病原学、流行病学以及制定疫苗生产策略提供重要信息。     

东北三省6株肾综合征出血热病毒生物学性状的研究

肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)是我国重点防治的急性传染病,对于流行于不同地区的汉坦病毒(hanatavirus)进行分离和系统地鉴定,可以为HFRS的防治提供科学依据,特别是对流行株血清型及基因型的确定,是研制疫苗和确定防治重点及对策的重要前提,也为研究我国汉坦病毒的遗传和进化特征奠定了基础。研究对6株流行于东北三省的汉坦病毒进行了全面鉴定,其中H8205、H8207株为人源性汉坦病毒,其余均为鼠源性汉坦病毒。研究表明,6株病毒具有病毒型别明确,抗原性较为广谱,免疫原性好,在Vero细胞上适应能力强及病毒滴度高,以及来源及传代历史清楚等特点,适合作为双价HFRS纯化灭活疫苗候选毒株,并且可建立起完善和合格的生产用毒种库,为我国生产以Vero细胞为基质的双价肾综合征出血热纯化灭活疫苗奠定了坚实的基础。     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

汉坦病毒H8205部分核壳蛋白基因在E.coli中的表达

根据汉坦病毒H8205株NP基因的序列,设计一对引物,扩增NP前292个氨基酸多肽基因片段,克隆于表达载体pGEX-3X,与载体中26kD的谷胱苷肽巯基转移酶(GST)融合表达.SDS-PAGE显示表达产物(GST-NP)主要以包涵体形式存在.Western-blotting表明此融合蛋白有抗原性.包涵体经分离、洗涤、溶解后,Sepharose 6B层析纯化,用此融合蛋白作抗原,进行ELISA法检测临床HFRS病人标本的IgG和IgM,有很好的特异性和敏感性.有生物活性的汉坦病毒H8205 NP的体外表达成功,为汉坦病毒基因工程抗原的大量制备奠定了基础,也为汉坦病毒的临床检测和流行病学调查提供了一种廉价、安全、可靠的抗原.     

汉坦病毒基因变异研究进展

自1976年韩国学者Lee从黑线姬鼠肺组织中分离到汉坦病毒(HV)以来,迄今已确立了16种血清型/基因型的HV,且不断发现新的毒株,如日本的Tobetsu病毒[1],波利维亚的Rio Mamore病毒[2]和西伯利亚的Topografov病毒[3].HV与其它由节肢动物传播的布尼亚病毒科病毒不同,它由特殊的啮齿类或食虫类动物作为其储存宿主,目前仅有5%的啮齿类动物检测到HV[4],这提示未来可能会发现新的毒株类型.     

汉坦病毒东方田鼠分离株ZT10的M基因序列的特性分析

分析东方田鼠分离汉坦病毒ZT10株M基因分子特征。提取汉坦病毒ZT10株感染细胞的总RNA,应用逆转录聚合酶链反应(RT-PCR)扩增ZT10株M片段全基因,克隆于T载体并测序,对其进行序列分析。结果显示汉坦病毒ZT10株的基因组M片段长度为3651个核苷酸,编码1133个氨基酸。序列分析表明其为Seoul型汉坦病毒。与八株Seoul型汉坦病毒的M片段同源性为84.0%～96.3%,而与HTN型汉坦病毒的同源性则较低,与从田鼠分离的汉坦病毒(ProspectHillvirus,Tulavirus,Khabarovskvirus,Islavistavirus)核苷酸同源性仅为57.5%～60.9%,且与浙江省Seoul型分离株Guo3同源性较低,表明浙江省可能存在着另一Seoul亚型的汉坦病毒。     

汉坦病毒东方田鼠分离株ZT10的M基因序列的特性分析

分析东方田鼠分离汉坦病毒ZT10株M基因分子特征.提取汉坦病毒ZT10株感染细胞的总RNA,应用逆转录聚合酶链反应(RT-PCR)扩增ZT10株M片段全基因,克隆于T载体并测序,对其进行序列分析.结果显示汉坦病毒ZT10株的基因组M片段长度为3651个核苷酸,编码1133个氨基酸.序列分析表明其为Seoul型汉坦病毒.与八株Seoul型汉坦病毒的M片段同源性为84.0%～96.3%,而与HTN型汉坦病毒的同源性则较低,与从田鼠分离的汉坦病毒(Prospect Hill virus, Tula virus, Khabarovsk virus, Isla vista virus)核苷酸同源性仅为57.5%～60.9%,且与浙江省Seoul型分离株Guo3同源性较低,表明浙江省可能存在着另一Seoul亚型的汉坦病毒.     

4株鸡马立克氏病病毒国内分离株Meq基因的克隆与序列分析

根据鸡马立克氏病病毒(MDV)GA株Meq基因序列,设计并合成一对用于扩增Meq基因的引物,利用这对引物通过PCR方法分别扩增4株东北地区分离的强毒株、国内标准强毒株J-1株、国内疫苗株814株的Meq基因片段,进行克隆测序,对4株MDV分离毒株Meq基因与国内传统毒株Meq基因及GenBank上收录的国内外9株毒株Meq基因序列进行比较分析.序列比较显示,不同的MDV株的Meq基因序列相对比较保守,它们相互间氨基酸序列的同源性在96.5%～99.7%之间.4株MDV分离毒株Meq基因在相关报道中提到的与毒力相关的脯氨酸重复区存在点突变;3株分离毒株Meq基因上相同位置均存在两个定点突变,这两处点突变是国内近几年分离株所特有的,国外已发表的MDV毒株Meq序列中不存在这种变化.分离株Meq基因的这些突变和毒株毒力的关系具有一定的规律性,但是这些规律性还有待进一步研究.     

Detection and characterization of three zoonotic viruses in wild rodents and shrews from Shenzhen city,China

Diverse species of rodents and shrews, which are abundant worldwide, harbor a variety of viruses;some of these are closely related to human viruses and possess zoonotic potential. Previously studies have demonstrated that the mammarenavirus and hantavirus carried by rodents or shrews could cause diseases in human population. To determine the distribution of zoonotic viruses in Shenzhen city, the major city in southern China with a high population density, we analyzed 225 rodents(Rattus norvegicus and Rattus flavipectus) and 196 shrews(Suncus murinus) from urban and rural districts for the presence of mammarenavirus, hantavirus, and hepatitis E virus(HEV) by RT-PCR targeting the conserved regions. The infection rates for mammarenavirus, hantaviruses,and HEV in rodents and shrews were 3.56%, 6.89%, and 1.66%, respectively. Partial genome fragment analysis indicated that mammarenavirus and hantavirus strains had more than 90% and 99% nucleic acid identity with Cardamones virus and Seoul virus, respectively, which cause diseases in humans. Although the present HEV strains identified are typically found worldwide,phylogenetic analysis demonstrated a divergence of 16%. To our knowledge, the present work is the first report of the prevalence of mammarenavirus, hantaviruses, and rat HEV strains in rodents and shrews from Shenzhen city, China. Our findings highlight the zoonotic potential of rodent-and shrew-borne mammarenavirus and hantavirus, and the biodiversity of rat HEV isolates in Shenzhen city. The present work suggests that utilization of good hygiene habits is important to minimize the risk of zoonosis.     

Analysis of hantavirus genetic diversity in Argentina: S segment-derived phylogeny

Nucleotide sequences were determined for the complete S genome segments of the six distinct hantavirus genotypes from Argentina and for two cell culture-isolated Andes virus strains from Chile. Phylogenetic analysis indicates that, although divergent from each other, all Argentinian hantavirus genotypes group together and form a novel phylogenetic clade with the Andes virus. The previously characterized South American hantaviruses Laguna Negra virus and Rio Mamore virus make up another clade that originates from the same ancestral node as the Argentinian/Chilean viruses. Within the clade of Argentinian/Chilean viruses, three subclades can be defined, although the branching order is somewhat obscure. These are made of (i) "Lechiguanas-like" virus genotypes, (ii) Maciel virus and Pergamino virus genotypes, and (iii) strains of the Andes virus. Two hantavirus genotypes from Brazil, Araraquara and Castello dos Sonhos, were found to group with Maciel virus and Andes virus, respectively. The nucleocapsid protein amino acid sequence variability among the members of the Argentinian/Chilean clade does not exceed 5.8%. It is especially low (3.5%) among oryzomyine species-associated virus genotypes, suggesting recent divergence from the common ancestor. Interestingly, the Maciel and Pergamino viruses fit well with the rest of the clade although their hosts are akodontine rodents. Taken together, these data suggest that under conditions in which potential hosts display a high level of genetic diversity and are sympatric, host switching may play a prominent role in establishing hantavirus genetic diversity. However, cospeciation still remains the dominant factor in the evolution of hantaviruses.     

2002～2004年浙江省无菌性脑膜炎暴发疫情的病原学研究

2002～2004年浙江省发生了无菌性脑膜炎暴发疫情,为了及时查明病因,分析病原的分子特征并进行病原溯源,我们采集患者脑脊液和粪便样本271份,用RD和Hep-2细胞同时分离病毒,对分离株VP1和VP4/VP2基因测序,进行同源性与进化分析。结果从271份样本中分离到埃柯病毒30型(E30)78株;对31株分离株VP1区核苷酸(nt)序列测定,其长度均为876nt,推导编码292个氨基酸(aa)。浙江E30株与原型株Bastianni在VP1区的nt和aa同源性分别为84.7%～86.3%和92.1%～94.2%;浙江E30株之间nt和aa的同源性分别为87.1%～99.4%和96.2%～100%。在VP1基因进化树上浙江E30株分别位于G和H基因亚型分支上,与浙江E30G亚型株亲缘关系最近的国内外毒株分别为2003年江苏、山东株和1999年乌克兰株;与浙江E30H亚型株亲缘关系最近的毒株为2008年韩国株。VP4/VP2区同源性与进化分析结果与VP1相似。结果表明2002～2004年浙江省无菌性脑膜炎暴发疫情由E30G和H二类不同基因亚型流行株引起;H基因亚型株推测为新的E30变异株,首先分离于2002年浙江省。     

Hantaviruses: an emerging public health threat in India? A review

The emerging viral diseases haemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) are a cause of global concern as they are increasingly reported from newer regions of the world. The hantavirus species causing HFRS include Hantaan virus, Seoul virus, Puumala virus, and Dobrava-Belgrade virus while Sin Nombre virus was responsible for the 1993 outbreak of HCPS in the Four Corners Region of the US. Humans are accidental hosts and get infected by aerosols generated from contaminated urine, feces and saliva of infected rodents. Rodents are the natural hosts of these viruses and develop persistent infection. Human to human infections are rare and the evolution of the virus depends largely on that of the rodent host. The first hantavirus isolate to be cultured, Thottapalayam virus, is the only indigenous isolate from India, isolated from an insectivore in 1964 in Vellore, South India. Research on hantaviruses in India has been slow but steady since 2005. Serological investigation of patients with pyrexic illness revealed presence of anti-hantavirus IgM antibodies in 14.7% of them. The seropositivity of hantavirus infections in the general population is about 4% and people who live and work in close proximity with rodents have a greater risk of acquiring hantavirus infections. Molecular and serological evidence of hantavirus infections in rodents and man has also been documented in this country. The present review on hantaviruses is to increase awareness of these emerging pathogens and the threats they pose to the public health system.     

Hantavirus-specific antibodies in rodents and humans living in Kuwait

Hantaviruses are found in widely scattered areas of the world and are transmitted by inhalation of virus-contaminated aerosols of rodent excreta. The present study was undertaken in Kuwait to investigate the serological evidence for hantavirus infection in rodents and humans. Sera were collected from 283 wild rodents and 183 human subjects (46 Kuwaitis and 137 non-Kuwaitis). The rodent sera were investigated for the presence of antibodies against the Seoul and Puumala strains of the hantaviruses by enzyme-linked immunosorbent assay and immunofluorescence technique using the virus-infected Vero E6 cells. The findings showed the presence of anti-hantavirus antibodies in seven out of the 283 (2.8%) rodents. Antibodies against the Seoul strain were present in six (2.1%) and against the Puumala strain in three (1%) rodents. Further, it was observed that three out of 84 (3.6%) of the Rattus norvegicus and four out of 174 (2.3%) Mus musculus had anti-hantavirus antibodies. Two rodents belonging to species Mus musculus had antibodies against both strains of the hantaviruses. Out of 183 human sera, 13 (7%) were positive for hantavirus antibodies. Among the Kuwaitis 5/46 (11%) and among the non-Kuwaitis 8/137 (6%) were positive for the hantavirus antibodies. Antibodies to both Puumala and Hantaan strains were detected in Kuwaitis as well as in non-Kuwaitis. Although no human case of hantavirus illness has yet been reported in Kuwait, the serological evidence of infection suggests a constant vigil.     

我国分离的XJ-90260病毒鉴定为西方马脑炎病毒

XJ－90260病毒是从新疆乌苏县境内采集的赫坎按蚊中分离到的一株病毒,病毒的鉴定结果显示：XJ－90260病毒可引起BHK－21细胞病变,表现为圆缩,脱落;可引起Vero细胞病变,表现为圆缩,破碎,脱落;可以在C6／36细胞中增殖,但不引起细胞病变。对3日龄小白鼠2－3天致死,对3周龄小白鼠3－4天致死。该病毒株对酸、乙醚敏感,抵抗5－氟脱氧尿苷。病毒与甲病毒组特异性免疫腹水起反应,与乙型脑炎病毒及布尼亚病毒组特异性免疫腹水不反应。进一步的分子生物学鉴定表明,该毒株基因组3′非编码区（ntranslated region,UTR）核苷酸序列具有典型的西方马脑炎病毒特征,与标准西方马脑炎病毒的首次报导,有重要的流行病学意义。我国9省区,886份血清的流行病学调查显示,该病毒抗体阳性血清24份,阳性率为2.71%。其中新疆（8／157）,河南（6／76）、甘肃（5／94）三省区抗体阳性数较多,占总阳性数的79.2%(19/24)。     

Prevalence of antibody to hantaviruses in humans and rodents in the Caribbean region of Colombia determined using Araraquara and Maciel virus antigens

We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV) or the N protein of Araraquara virus (ARAV) as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5%) and anti-ARAV antibodies in 21 sera (7.34%). Of the 10 samples that were positive for MACV, seven (70%) were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4%) were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis), six Heteromys anomalus (Heteromyidae), one Proechimys sp. (Echimyidae) and 105 Muridae (34 Rattus rattus and 71 Mus musculus). All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.     

2009年云南省边境地区健康儿童肠道病毒测序定型和ECHO7、ECHO13病毒的基因特征分析

了解2009年缅甸入境健康儿童和云南省口岸地区健康儿童肠道病毒(enterovirus,EV)带毒情况并对埃柯病毒7型(ECHO7)和埃柯病毒13型(ECHO13)的基因特征进行了描述。采集9个边境口岸小于15岁的健康儿童粪便标本271份,进行病毒分离和基因测序定型。271份便标本中共检测到EV30株(带毒率为11.1%),其中脊髓灰质炎病毒(poliovirus,PV)6株(阳性率2.8%),均为疫苗株,未发现脊灰野病毒;检测到非脊灰病毒(NPV)24株(阳性率8.9%)。经VP1区核苷酸序列测定,24株NPV全部为人类肠道病毒B组(HEV-B,6个血清型),其中13株为埃柯病毒7型(echovirus 7,ECHO7,占54.17%),5株为ECHO13(占20.83%)。未分离到HEV-A组,HEV-C组和HEV-D组病毒。2009年缅甸入境健康儿童和云南省口岸地区健康儿童中肠道病毒携带率较高,且均为HEV-B组病毒,其中主要型别为ECHO7和ECHO13,这两种病毒存在基因多样性特点(即存在不同的基因型)。     

Serological survey for viral pathogens in Turkish rodents

Wild rodents (n = 330) were trapped around the villages of Altindere and Co?andere (Ma?ka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihem?in, Rize Province), and Bozdag (Odemi?, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.     

Mathematical Models for Hantavirus Infection in Rodents

Hantavirus pulmonary syndrome is an emerging disease of humans that is carried by wild rodents. Humans are usually exposed to the virus through geographically isolated outbreaks. The driving forces behind these outbreaks is poorly understood. Certainly, one key driver of the emergence of these viruses is the virus population dynamics within the rodent population. Two new mathematical models for hantavirus infection in rodents are formulated and studied. The new models include the dynamics of susceptible, exposed, infective, and recovered male and female rodents. The first model is a system of ordinary differential equations while the second model is a system of stochastic differential equations. These new models capture some of the realistic dynamics of the male/female rodent hantavirus interaction: higher seroprevalence in males and variability in seroprevalence levels.