深圳市肾综合征出血热患者汉坦病毒基因特征研究

目的 研究深圳市引起肾综合征出血热(HFRS)的汉坦病毒主要型别及分子特征,为该市汉坦病毒的防制提供依据.方法 收集各医院送检HFRS患者急性期血清标本,提取血清中病毒RNA作为模板,采用逆转录-套式PCR扩增汉坦病毒基因组M片段G2区基因并进行序列测定,对汉坦病毒进行基因分型和同源性分析.结果 深圳市HFRS病例男性多于女性,患者发病年龄主要集中在20-60岁,其中20-49岁病例数最多,共发病242例,占发病总数的85.82％.从282例标本中用HTN型特异性引物扩增阳性4例,占1.42％;用SEO型特异性引物扩增阳性50例,占17.73％;总阳性率为19.15％.序列比较分析发现,深圳地区流行的SEO型汉坦病毒核苷酸序列的差异相对较小,其基因的离散度为0％-4.9％.从种系发生树看这些病毒分布在一个分支上,均为S2亚型.HTN型汉坦病毒的变异率较高,其基因的离散度为6.7％-21.0％,二例为H7亚型,二例为H9亚型,均为深圳市首次报道.结论 不同年份引起深圳地区HFRS的汉坦病毒仍以SEO型为主,且病毒变异较小,稳定性较高.首次发现HTN型病例存在.结合流行病学调查资料,证实HTN病例均为输入性病例.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

河北省鼠携带汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省肾综合征出血热(HFRS)疫源地宿主动物所携带汉坦病毒的基因型和基因亚型.方法 根据76-118株和R22株的保守序列设计型特异性引物,用RT-nested PCR从IFA筛选的阳性鼠肺中扩增部分M片段,经琼脂糖凝胶电泳进行基因分型,并从中选取部分扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 32份经IFA筛选阳性的鼠肺标本经RT-nested PCR扩增出418bp特异性目的 片段,均为SEO型.10份扩增产物测序结果 表明G2片段变异不大,同源性高达98.0%～100%,与R22株和76-118株的同源性分别为93.3%～94.3%和67.7%～69.0%.结论 依据G2片段,河北省HFRS疫源地汉坦病毒基因型以SEO型病毒为主,主要为S3亚型,同亚型病毒基因高度同源,遗传物质相对稳定.不同鼠种可携带同亚型汉坦病毒.     

Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes.

Worldwide, hantaviruses cause more than 100,000 human infections annually. Rapid and accurate methods are important both in monitoring acute infections and for epidemiological studies. We and others have shown that the amino termini of hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease. Accordingly, we expressed truncated Ns (amino acids 1 to 117) in Escherichia coli from the five hantaviruses known to be pathogenic to man; Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN), and Puumala (PUU) viruses. In order to obtain pure antigens for use in an enzyme-linked immunosorbent assay (ELISA), the recombinant proteins were purified by polyhistidine-metal chelate affinity chromatography. Polyclonal animal antisera and a panel of serum specimens from hantavirus-infected individuals from Scandinavia, Slovenia, Russia, Korea, China, and the United States were used to evaluate the usefulness of the method. With both human and animal sera, it was possible to designate the antibody response into two groups: those with HTN, SEO, and DOB virus reactivity on the one hand and those with SN and PUU virus reactivity on the other. In sera from Scandinavia, European Russia, and the United States, the antibody response was directed mainly to the PUU and SN virus group. The sera from Asia reacted almost exclusively with the HTN, SEO, and DOB types of viruses. This was true for both the immunoglobulin M (IgM) and IgG antibody responses, indicating that this type of discrimination can be done during the acute phase of hantavirus infections. Both the HTN, SEO, and DOB virus and the PUU and SN virus types of antibody response patterns were found in patients from the Balkan region (Solvenia).     

汉城病毒新亚型的发现

目的 至今为止,汉城病毒间核苷酸序列的同源性均在９５％以上,而氨基酸序列显示出更高的保守性。本研究对一株分离子褐家鼠的汉城病毒Ｇｏｕ３株的Ｍ、Ｓ片段进行了部分核苷酸序列测定,观察其与已知汉城病毒的差异。方法 采用逆转录－ＰＣＲ方法对Ｇｏｕ３病毒进行了扩增鉴定,并对其部分Ｍ和Ｓ基因组片段进行了碱基序列测定。结果 表明Ｇｏｕ３与其他ＳＥＯ病毒有不同的ＰＣＲ扩增特点。序列测定也显示其是一个差异较大的汉城病毒株,与基佗汉城型病毒的同源性却只有８５％左右。但由其推导出的Ｇｏｕ３的部分糖蛋白的氨基酸序列与其他汉城型病毒相比却高度保守。系统发生树分析表明Ｇｏｕ３是介于汉城病毒与汉滩病毒之间,但其亲缘关系较近于已知汉城病毒。结论 Ｇｏｕ３病毒为汉城病毒,是属于汉城病毒一个新亚型。     

浙江东方田鼠分离汉坦病毒株S基因的克隆和序列分析

目的对国内首次从东方田鼠分离到的汉坦病毒（HV）ZT10S基因克隆进行序列分析。方法参考GenBank发表的汉坦病毒核蛋白基因序列，设计合成一对引物，提取分离株ZT10细胞培养物的总RNA，对ZT10S基因进行RT-PCR扩增，产物经琼脂糖电泳分析，呈现一条约1．7kb的条带，回收纯化后，将其克隆至pGEM．T载体中，然后进行核苷酸序列的测定及分析。结果与4株SEO型病毒（SR-11、R22、Gu03、8610）核苷酸和氨基酸同源性分别为87．9％～96．0％和96．9％～97．9％，与HTN型病毒株76-118的核苷酸和氨基酸同源性分别为71．3％和81．9％，与其他型汉坦病毒的同源性均很低，表明此分离株ZT10为SEO型汉坦病毒。结论为进一步研究其进化和变异提供了有利条件，也为生产SEO型汉坦病毒特异性核蛋白抗原，免疫血清学诊断试剂的制备和分子生物学研究奠定了基础。     

河北省人感染汉坦病毒G2片段基因分型及序列特征分析

目的 了解河北省人感染汉坦病毒的基因型和基因亚型.方法 根据76～118株和R22株的C2编码区设计型特异性引物,利用RT-nested PCR技术对采集的HFRS患者中汉坦病毒抗体阳性的血清标本进行检测及基因分型,从中选取部分标本的扩增产物纯化回收后进行核苷酸序列测定,用DNAStar软件包进行基因分析.结果 69份阳性血清标本经RT-nested PCR检测17份阳性,检出率为24.64%,其中≤7 d的标本检出率为34.29%,8～14 d的检出率为19.23%,≥14 d的未能检出.17份阳性标本均为SEO型.对其中9份扩增产物的G2区测序后表明,9份血清标本的同源性为92.0%～100.0%,其中HeB7与其他8份标本的同源性为92.0%～95.0%,属不同亚型,与R22的同源性为97.7%,同属于S1亚型;除HeB7外其余8份标本同源性高达95.7%～100%,同属于S3亚型.9份标本与76～118株的同源性仅为70.3%～72.7%.结论 河北省人感染汉坦病毒以SEO型为主,亚型以S3为主,也存在S1亚型.在一定范围内,同型汉坦病毒基因相对保守,具有区域稳定性特征.     

Low pH-induced cytopathic effect--a survey of seven hantavirus strains.

Hantaviruses do not produce cytopathic effects (CPE) in cell culture. However, a syncytial CPE can be induced in 7-day cultures of hantavirus growing in Vero E6 cells by reduction of the pH to approximately 6.2 using a HEPES based buffer. The appearance of this acid induced CPE was examined for seven different hantavirus strains. The differences noted were striking and reflected the taxonomic differences between hantaviruses. At 10-100 TCID50% the size of syncytial foci was very large for Seoul type viruses and smallest for Puumala viruses. The size of syncytia for Hantaan (HTN) virus was intermediate between Puumala (PUU) and Seoul (SEO) type viruses.     

Distribution of hantavirus serotypes Hantaan and Seoul causing hemorrhagic fever with renal syndrome and identification by hemagglutination inhibition assay.

An epidemiologic evaluation of patients with hemorrhagic fever with renal syndrome from different locations in the People's Republic of China was conducted to define the prevalence of two Hantavirus serotypes, Seoul (SEO) and Hantaan (HTN). Serum specimens were collected between 5 and 14 days after the onset of illness and were tested for antibodies by both hemagglutination inhibition (HI) and plaque reduction neutralization (PRN). By the HI test, the geometric mean titer (GMT) of antibodies to SEO in the sera from individuals from Kaifeng City of Henan Province was five times higher than that to HTN. In contrast, by the HI test, the sera from individuals from Jiande County of Zhejiang Province had a GMT of antibodies to HTN that was seven times higher than that to SEO. In the sera from individuals from Shanghai, only a twofold difference was observed in HI antibody titers to the two hemagglutinins by the HI test, with that to HTN being higher than that to SEO. By the PRN test, the GMT ratios of antibody between HTN and SEO strains from individuals in Kaifeng, Jiande, and Shanghai were found to be 1:13, 14:1, and 2:1 respectively. A close correlation (r = 0.8219) and concordance rate (78.3%) were observed between the PRN and HI tests for the identification of the serotypes of individual cases of hemorrhagic fever with renal syndrome. The hantavirus serotypes from individuals in Kaifeng and Jiande were identified as predominantly SEO and HTN, respectively, and those from individuals in Shanghai had an indeterminant serotype defined by these two techniques. The HI test appears to be a simple and reliable way of determining the predominant hantavirus that causes HFRS in a given geographic area.     

Characterization of truncated hantavirus nucleocapsid proteins and their application for serotyping

Hemorrhagic fever with renal syndrome (HFRS) is a fulminant infectious disease characterized by fever, hemorrhage, renal impairment, and thrombocytopenia. Hantaviruses associated with this belong to different serotypes: Hantaan (HTN), Seoul (SEO), Dobrava/Belgrade (DOB), and Puumala (PUU). The first two, HTN and SEO, are endemic in China. To investigate the epidemiology of HFRS and virus transmission in China, we constructed prokaryotic plasmids encoding truncated recombinant HTN and SEO nucleocapsid proteins (NPs), which lacked 154 amino acid (aa), 99 aa, or 49 aa in the N-terminal region, respectively. After expression, the truncated rNPs were tested as serotyping antigens, particularly for use in the enzyme-linked immunosorbent assay (ELISA). In addition, 68 acute and 52 convalescent sera were collected from HFRS patients from Harbin, Lantian, and Kaifeng regions in China in 2004, which had hantavirus specific antibodies by IFA. A neutralization test was used to differentiate these, which showed that 73 were due to HTN infection, 33 to SEO infection, and 14 undetermined. By ELISA, the truncated rNPs, that lacked 99 (rNP100) or 49 (rNP50) N-terminal amino acids of the NPs of HTN and SEO, were able to differentiate HTNV and SEOV-specific immune sera, but the rNP155 could not. Particularly, the ELISAs based on the rNP50s had a result comparable to PRNT. Thus, the rNP50 is recommended as efficient serotyping antigen for hantavirus infection diagnosis by ELISA.     

Genetic properties of medium (M) and small (S) genomic RNA segments of Seoul hantavirus isolated from <Emphasis Type="Italic">Rattus norvegicus</Emphasis> and antigenicity analysis of recombinant nucleocapsid protein

A novel isolate of Seoul (SEO) hantaviruses was detected and identified in Rattus norvegicus in Shandong Province, China and designated as JUN5-14. The partial M segment and the coding region of nucleocapsid protein (NP) in the S segment of JUN5-14 were PCR-amplified and sequenced. Nucleotide sequence analysis of the partial M segment (300 bp) revealed that JUN5-14 isolate was closely related to former SEO isolates in Shandong (97.3–99.0% homology) and non-Shandong SEO viruses (84.1–97.7% homology) but distantly related to other hantaviruses (61.5–75.1% homology). Consistent with the M segment, the coding region of the NP showed 87.5–97.8% and 97.9–99.8% identity with SEO viruses and 55.2–75.8% and 47.2–84.4% homology with other hantaviruses, at nucleotide and amino acid level, respectively. The virus isolate was identified as a member of the subtype 3 (S3) of SEO viruses by phylogenetic trees generated from the nucleotide sequences of the S and M segments. In order to develop a diagnostic assay for hantavirus infection in human, the full-length NP gene of JUN5-14 was expressed in BHK21 cells using the T7 RNA polymerase expression system. The NP expression was confirmed by indirect immunofluorescence assay (IFA) and Western blotting. The expressed NP protein was used as antigen to detect antibody response against NP in patients with hemorrhagic fever with renal syndrome (HFRS) in an IgG-IFA. Sixteen out of seventeen serum samples showed positive for the presence of anti-NP antibodies, indicating that the recombinant NP (rNP) protein of JUN5-14 was a good antigen for detecting hantavirus infection in human. This work was supported by Key Scientific and Technological Innovative Research Plan of Shandong Province (No. CX02102), China and the Scientific Foundation of Innovative Research Team in Shandong University, China.