Diversity and Distribution of Host Animal Species of Hantavirus and Risk to Human Health in Jiuhua Mountain Area,China

Objective To investigate the diversity and the distribution of host animal species of hantavirus and the effect on human health in Jiuhua Mountain area,China.Methods The host animal species of hantavirus was surveyed by using the trap method and the species diversity was evaluated by using the Simpson,Shannon-Weaner,and Pielou indices.Hantavirus antigens or antibodies in lung and blood samples of all the captured host animals were detected by direct or indirect immunofluorescence.Results Nine animal species of hantavirus were distributed in the forest ecosystem of Jiuhua Mountain.Of these,Niviventer confucianus and Apodemus agrarius were predominant,and N.confucianus,Rattus norvegicus,and Mus musculus had relatively large niche breadth index values.The host animals in the eastern and western mountain regions shared similar biodiversity index characteristics,predominant species,and species structures.Hantavirus was detected in 5 host animal species in Jiuhua Mountain area,the carriage rate of hantavirus was 6.03%.The average density of host animals in forest areas of the mountainous area was only 2.20%,and the virus infection rate in the healthy population was 2.33%.Conclusion The circulation of hantavirus was low in the forest areas of Jiuhua Mountain and did not pose a threat to human health.

Stable Expression of Hantavirus H8205 Strain G1/IL-2 Gene and Immune Protection of the Fusion Gene

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE elec- trophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly （i.m.） three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies （NAb） were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1：20-1：80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

Preparation and antigenic characterization of rat monoclonal antibodies againts Hantavirus

Monoclonal antibodies (McAb) to Hantavirus (HTV)were derived from the hybridoma fused with Lou/c plasmocytoma rat cells (IR983F) and spleen cells from lou/c inbred rat immunized with HTV Chen strain. Eleven cell lines producing monoclonal antibodies direc

First Evidence of Hantavirus in Central Iran as an Emerging Viral Disease

Hantavirus is a zoonosis transmitted from rodents to humans. Asymptomatic infected rodents can secrete hantaviruses in the urine, feces, and saliva. The main route of infection transmission to human is aerosols contaminated with the virus. This study was designed to evaluate the serological and molecular prevalence of hantavirus as an emerging zoonoses disease among street sweepers in Isfahan province, central Iran. Serum samples from 200 street sweepers in healthy condition and those with recent renal failure were tested by ELISA (IgM and IgG). Molecular analysis was subsequently applied for IgM positive cases. From these samples, 9 (4.5%) were positive, of which 2 (22.22%) were positive for both IgM and RT-PCR, while 7 (77.77%) were positive for IgG. The mean age and work experience of the positive cases were 39.7 and 11.5 respectively. According to our observations, all positive cases reported prevalence of rodents in their work place. The logistic regression test showed that the age and work experience were not risk factors for being positive, but prevalence of rodents in work place was a risk factor for being positive, when compared with negative cases. This is the first comprehensive study on the prevalence of hantavirus with positive results coming from Iranian population, which can raise the public awareness for the hantavirus infections as a public health threat.

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections

BACKGROUND Serologic cross-reactivity between hantaviruses often complicates the interpretation of the results.AIM To analyze the diagnostic value of indirect immunofluorescence assay(IFA)and western blot(WB)in the diagnosis of hantavirus infections.METHODS One hundred eighty-eight serum samples from Puumala(PUUV)and Dobrava(DOBV)orthohantavirus infected patients were analyzed.Serology was performed using commercial tests(Euroimmun,Lübeck,Germany).RESULTS Using IFA,49.5%of acute-phase samples showed a monotypic response to PUUV,while 50.5% cross-reacted with other hantaviruses.The overall cross-reactivity was higher for immunoglobulin G(IgG)(50.0%)than for immunoglobulin M(IgM)(25.5%).PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan(12.3%/31.5%),Seoul(7.5%/17.8%),DOBV(5.4%/28.1%),and Saaremaa(4.8%/15.7%).Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan(76.2%/95.2%),Saaremaa(80.9%/83.3%),and Seoul(78.6%/85.7%)and moderate with PUUV(28.5%/38.1%).Using a WB,serotyping was successful in most cross-reactive samples(89.5%).CONCLUSION The presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections,confirming serotype in most IFA cross-reactive samples.

Hantavirus infection:a global zoonotic challenge

Hantaviruses are comprised of tri-segmented negative sense single-stranded RNA, and are members of the Bunyaviridae family. Hantaviruses are distributed worldwide and are important zoonotic pathogens that can have severe adverse effects in humans. They are naturally maintained in specific reservoir hosts without inducing symptomatic infection. In humans, however,hantaviruses often cause two acute febrile diseases, hemorrhagic fever with renal syndrome(HFRS) and hantavirus cardiopulmonary syndrome(HCPS). In this paper, we review the epidemiology and epizootiology of hantavirus infections worldwide.

Nucleotide sequence characterization and phylogenetic analysis of hantaviruses isolated in Shandong Province,China

Background China is the most severe endemic area of hemorrhagic fever with renal syndrome （HFRS） in the world with 30 000-50 000 cases reported annually, which accounts for more than 90% of total number of cases worldwide. The incidence rate of the syndrome in Shandong Province is one of the highest in China, which has ever reached 50 per 100 000 persons per year. However, the molecular characteristics of hantaviruses （HV） epidemic in Shandong Province remain unclear. Therefore it is useful to clarify nucleotide sequence and phylogenetic characteristics of HV isolated in Shandong Province in order to provide better advices to control and prevent HFRS. Methods RNAs were extracted from sera of clinically diagnosed patients and positive rodent lungs that were detected by indirect immunofluorescent assay （IFA）. Partial M segments of HV were amplified from the RNAs with reverse transcription nested polymerase chain reactions （nested PCR） using hantavirus genotype specific primers. The nested PCR products were sequenced and compared with those from previously epidemic isolates in Shandong and with other representative HV sequences from GenBank. Phylogenetic tree analyses were performed based on the sequences of the M genes. Results Thirty-four HV isolates in Shandong showed 67.1%-100% nucleotide identities. The nucleotide homologies among 6 Hantaan viruses （HTNV） isolates in Shandong were 78.1%-98.7%, while the homologies among 28 Seoul virus （SEOV） isolates in Shandong were 93.7%-100%. There were at least 3 subtypes HTNV （H2, H5, H9） and 2 subtypes SEOV （S2, S3） in Shandong Province. Conclusions In Shandong Province, the homologies of HTNV were lower and there were no predominant subtypes, while the homologies of SEOV were higher and S3 was the predominant subtype. The homologies of SEOV from rodents were higher than those from patients. The distribution of subtypes in Shandong was similar to that of the adjoining provinces. Phylogenetic analyses of the sequences showed geographic clustering of HV in Shandong.

Enhancing DNA vaccine potency against hantavirus by co-administration of interleukin-12 expression vector as a genetic adjuvant

Background The heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development Of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus. Methods BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleoeapsid protein alone or in combination with a plasmid expressing murine interleukin-12 (pcIL-12). Booster immunizations were employed 2 times at 2-week interval. To evaluate the Immoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-gamma in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization. Results Antigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1: 50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3.1 + S alone, in which the mean titre was about 1:70. Interferon-gamma was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3.1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 coinjection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation. Conclusion Humoral and cytokine responses elicited by pcDNA3.1 + S inoculation can be modulated by coinoculation with pcIL-12 and efficiently induced Th1-dominant immune responses.

Hantavirus Infection during Pregnancy

Hantavirus infection is a global health challenge,causing widespread public concern.In recent years,cases of hantavirus infection in pregnant women have been reported in many countries.The infected pregnant women and their fetuses appear to have more severe clinical symptoms and worse clinical outcomes.Hence,to study the prevalence of hantavirus infection in pregnant women,this study will focus on the epidemiological distribution of the virus,different virus species penetrating the placental barrier,and factors affecting the incidence and clinical outcome of the infection in pregnant women and their fetuses.In addition,this review will also discuss the diagnostic tools and treatments for pregnant patients and provide an overview of the relevant future research.

Spatial structure of rodent populations and infection patterns of hantavirus in seven villages of Shandong Province from February 2006 to January 2007

Background Hemorrhagic fever with renal syndrome （HFRS） is endemic in Junan county, Shandong Province, China. We conducted geographic information system （GIS）-based spatial analysis with the objective of estimating the spatial distribution of rodent populations and their hantavirus infection patterns, to describe the spatial relationships of hantavirus strains in small ecological areas and to identify key areas in endemic areas of HFRS for future public health planning and resource allocation.

展望流行性出血热病原学研究

1976年在南朝鲜、1981年在我国相继成功地分离到流行性出血热病毒。这一突破性进展使我国也使全世界对这种疾病的研究和防治跨进一个新的阶段。

携带汉坦病毒宿主动物分布特征研究

目的研究西安地区携带汉坦病毒(HV)宿主种类及分布特点。方法夹夜法捕获宿主动物后鉴定种类,利用荧光定量逆转录聚合酶链反应(RT-PCR)技术检测宿主动物肺脏中HV核酸。结果 2011-2013年在监测地域共捕获动物2 577只,鼠密度为3.28%,在9月份达到5.6%;其中黑线姬鼠(Aa)占63.4%,小家鼠(Mm)和褐家鼠(Rn)分别占18.98%和12.88%;312份动物标本中检出HV核酸,其中Aa占88.78%,Rn占5.12%,Mm占4.49%,在黄胸鼠(Rf)和北小社鼩(Cs)也检出HV核酸,在8月和9月捕获的阳性宿主占到了全部阳性的56.73%,野外的阳性宿主占到了总阳性的87.82%。结论西安地区鼠密度在9月达到高峰,鼠种主要为Aa,Mm和Rn,携带HV的动物有Aa,Rn,Mm,Rf和Cs等,形成了在野外以Aa为主,居民区以Rn和Mm为主,阳性宿主动物主要出现在野外8月9月,这对本地区防控肾综合征出血热(HFRS)中宿主控制方面具有指导意义。

辽宁省鼠类感染肾综合征出血热的病原学及分子生物学检测比较

目的了解辽宁省鼠间带毒、鼠密度及鼠种构成与汉坦病毒的分子特征。方法采用夹夜法对鼠密度、鼠种构成及鼠带毒率进行监测,用间接免疫荧光方法检测HFRS抗原,用Vero-E6细胞对病毒进行传代分离,RT-PCR方法对阳性毒株进行基因分型及测序分析。结果鼠密度在春秋季各出现一个高峰,村内鼠密度高于村外鼠密度,褐家鼠为优势鼠种。带毒率无显著性差异,各鼠种间带毒率无显著性差异。5株病毒,有2个遗传分支,4株为HV的SEO型,1株为HTN型,4株SEO型HV基本位于系统发生树的同一进化枝上;1株HTN型HV,与汉滩病毒标准株76-118接近。结论辽宁省每年存在春秋季两个鼠密度高峰,带毒率同季节及鼠种无关。辽宁省是以SEO型HV为主的SEO和HTN混合型疫区,且SEO型汉坦病毒变异较小。

黑线姬鼠种群结构与携带汉坦病毒相关性

汉坦病毒(Hantavirus,HV)是肾综合症出血热(Hemorrhagic fever with renal syndrome,HFRS)的主要病原体之一,HV的主要宿主动物为黑线姬鼠(Apodemus agrarius)。针对西安市HFRS的持续高发病率,2010年7月—9月对西安市HFRS疫区捕获的110只黑线姬鼠(阳性62只)进行年龄、性别鉴定。通过病毒RNA提取分析,发现黑线姬鼠雌雄个体携带病毒无显著差异,但是不同年龄段黑线姬鼠携带汉坦病毒却具有显著差异,年龄结构与病毒携带具有极显著的相关性。

应用聚合酶链反应对我国不同来源肾综合征出血热病霉的型别分析

应用聚合酶链反应（ＰＣＲ），对分离自不同地区、不同宿主来源的２６株肾综合征出血热病毒进行了分型，其中包括４个型别的国际标准株，用异硫氰酸胍－酚－氯仿方法从感染的Ｖｅｒｏ－Ｅ６细胞中提取总ＲＮＡ，设计了５对寡核苷酸引物，一对为汉坦病毒特异性引物；４对为不同型特异性引物。ＰＣＲ分型表明，２６株中除１株Ｒｒ可同时被汉坦（ＨＹＮ）和Ｓｅｏｕｌ（ＳＥＯ）两型特异性引物扩增外，其余２５株分别只被４个型别引物中的一个所扩增，依次为ＨＴＮ１６株，ＳＥＯ７株，Ｐｕｕｍａｌａ（ＰＵＵ）１株，ＰｒｏｓｐｅｃｔＨｉｌｌ（ＰＨ）１株。ＰＣＲ分型的结果与空斑减少中和试验完全一致，表明ＰＣＲ可以对肾综合征出血热病毒准成分型。应用限制性内切酶分析了扩增产物，结果与理论基本一致，证实了扩增产物的特异性。

肾综合征出血热患者血清中汉坦病毒的检测及其序列分析

目的 为了进一步探讨RT -PCR用于HFRS临床标本中病毒基因检测的可行性及陕西西安地区近年主要流行汉坦病毒 (HV)毒株的分子流行病学特点。方法 本研究设计并合成了两套分别互补于HVM基因片段和S基因片段的引物 ,建立了检测HFRS患者临床血清标本中HV基因的RT -nestedPCR方法 ,并对扩增的部分HV靶基因进行了测序分析和比较。结果 119份HFRS病人血清 (经临床和IFA确诊 )经用S基因片段引物的RT -nestedPCR检测 ,阳性率分别为 :6 0 .5 % (<1w) ,34.1% (1～ 2w) ,4% (2～ 3w) ,0 % (>3w) ,31份HFRS急性期病人血清用M基因片段引物的RT -nestedPCR检测 ,仅 12份阳性 ,阳性率 40 %。结论 表明S基因片段引物的扩增效果优于M基因片段引物。 6份S基因片段引物扩增产物的测序结果显示 ,陕西西安地区HFRS感染的主要毒株为汉滩型 ,将PCR扩增的 6个靶基因序列与 76 - 118株S基因片段相应的核苷酸序列进行分析比较 ,同源性为 87.2 %～ 96 .0 % ;其推导的氨基酸序列与 76 - 118株核衣壳蛋白相应的氨基酸序列比较 ,同源性为 89.0 %～ 96 .0 %。

单价汉坦病毒灭活疫苗交叉免疫的中和抗体应答

目的：观察单价汉坦病毒灭活疫苗交叉免疫的中和抗体应答。方法：直接免疫酶斑减少中和试验、间接免疫荧光法。结果：在家兔进行单价汉坦病毒沙鼠肾细胞灭活疫苗二针基础免疫后１个月，再予另一型单价疫苗加强免疫，诱导较好的两型中和抗体应答，达到双价苗二针免疫效果；与初次免疫疫苗同型的中和抗体滴度亦显著提高。结论：汉坦病毒灭活疫苗存在交叉体液免疫反应；提示在已接受单价疫苗免疫的人群中进行交叉加强免疫，可能是有效、可靠、经济的免疫策略。

HFRS患者血清中HV-RNA的PCR检测及扩增产物的测序分析

目的 探讨 RT- PCR用于 HFRS临床标本中病毒基因检测的若干影响因素及西安地区近年主要流行汉坦病毒(HV)毒株的分子流行病学特点 .方法 设计合成了两套分别互补于 HV M基因片段和 S基因片段的引物 ,建立了检测HFRS患者临床血清标本中 HV基因的 RT- nested PCR方法 ,并对扩增的部分 HV靶基因进行了测序分析和比较 .结果 119份 HFRS患者血清 (经临床和 IFA确诊 )经用 S基因片段引物的 RT- nested PCR检测 ,阳性率分别为 :6 0 .5 %(<1wk) ,34 .1% (1～ 2 wk) ,4.0 % (2～ 3wk) ,0 % (>3wk ) ,31份 HFRS急性期患者血清用 M基因片段引物的RT- nested PCR检测 ,仅 12份阳性 ,阳性率 40 .0 % .将 PCR扩增的 6个靶基因序列与 76～ 118株 S基因片段相应的核苷酸序列进行分析比较 ,同源性为 87.2 %～ 96 .0 % ;其推导的氨基酸序列与 76～ 118株核衣壳蛋白相应的氨基酸序列比较 ,同源性为 89.0 %～ 96 .0 % .结论 S基因片段引物的PCR扩增效果优于 M基因片段引物 .6份 S基因片段引物扩增产物的测序结果显示 ,西安地区引起

聚合酶链反应技术用于汉坦病毒的基因分型

应用反转录ＰＣＲ技术对国内外分离的１８株汉坦病毒基因进行了检测，结果１０株血清Ⅰ型病毒仅能用Ⅰ型引物扩增，８株血清Ⅱ型病毒仅能用Ⅱ型引物扩增，与既往空斑减少中和试验的分型结果完全一致，两种ＰＣＲ产物经内引物再次扩增及酶切分析鉴定证明具有特异世，表明ＲＴ－ＰＣＲ是一项比较特异、敏感和简便实用的汉坦病毒基因分型方法。

云南省啮齿动物中汉坦病毒自然感染状况调查

目的调查云南省啮齿动物汉坦病毒自然感染状况，为制定云南省肾综合征出血热的预防控制提供科学依据。方法用鼠笼和鼠夹在云南省11个县（市）进行捕鼠，捕获的啮齿动物进行分类鉴定并解剖取肺脏组织和用直接免疫荧光法检测汉坦病毒。结果在调查的11个县（市）共捕获5属8种啮齿动物1303只，黄胸鼠和褐家鼠为居民区优势种，高山姬鼠和大绒鼠为野外优势种。调查县（市）汉坦病毒自然感染率范围为2、5％～7．1％；居民区黄胸鼠、褐家鼠、小家鼠和大足鼠中汉坦病毒自然感染率分别为3，8％（24／637）、5、5％（15／274）、4．2％（2／47）和3．0％（3／100）；野外高山姬鼠、大绒鼠、中华姬鼠和臭朐腈中汉坦病毒自然感染率分别为2．2％（3／139）、1．8％（3／163）、0（0／4）和14．3％（1／7）。结论云南省啮齿动物汉坦病毒自然感染普遍，存在以褐家鼠一黄胸鼠为主要宿主的汉城型病毒的循环形式和以高山姬鼠一大绒鼠为主要宿主的汉滩型病毒的循环形式。