汉坦病毒在Vero细胞上的适应传代及疫苗候选株的 …

目的 选育出在Ｖｅｒｏ细胞上繁殖力强、免疫原性和抗原性好的汉滩病及汉城病毒候选株。方法 国内外分离的１６株汉坦病毒（ＨＶ）在Ｖｅｒｏ细胞上进行连续终末稀释传代,研究各株的繁殖特性及传代后病毒滴度及抗原量的变化。结果 经过５次终末稀释传代,选育出Ｌ９９（汉城病毒）和８４ＦＬｉ（汉滩病毒）２株滴度高且稳定的病毒,在Ｖｅｒｏ细胞上具有良好和适应性,７～１０ｄ毒力接近峰值。ＥＬＩＳＡ测抗原量高峰则在１０～     

肾综合征出血热纯化疫苗候选毒株在Vero细胞上的适应传代及其免疫原性研究

目的 将肾综合征出血热纯化疫苗候选毒株适应于Vero细胞，并对其抗原性和免疫原性进行研究。方法 将汉滩病毒H8207株和汉城病毒Y86013株在Vero细胞上进行连续终末稀释传代，采用间接免疫荧光法、酶联免疫吸附试验和空斑减少中和试验，研究传代后毒株的繁殖特性、病毒滴度、抗原量及免疫原性。结果 经过5次终末稀释传代，两株病毒在Vero细胞上均显示出良好的适应性，从第六代开始病毒滴度稳定在7．0Lg TCID50/m1以上，第八代两毒株抗原量均已达到1：64，H8207株抗原量继续升高，最高时达1：256。用两株病毒不同培养代次上清液制备的单价原液灭活疫苗免疫家兔二针，免疫血清对同型毒株的中和效价均达到1：10。结论 两株病毒已适应于Vero细胞，且具有病毒滴度高和免疫原性良好的特性，适合用作Vero细胞肾综合征出血热灭活纯化疫苗的候选毒株。     

有源汉坦病毒疫苗候选株的分离鉴定及其某些特性 …

目的 对人源汉坦病毒疫苗候选株作分离鉴定及特性的研究。方法 用Ｖ ｅｒｏ细胞分离病毒,用免疫荧光法作病毒效价测定。用ＲＴ－ＰＣＲ及部分核苷酸序列分析等检定。结果 用Ｖｅｒｏ细胞从安徽省凤台县肾综合征出血热病人血清中分离一株汉坦病毒（ＨＶ）,其抗原特性及ＰＣＲ分型结果证明为汉滩病毒（Ⅰ型病毒）;其免疫血清对来源于不同地区的ＨＶ均有中和活性。Ｍ片段部分序列分析结果表明,该毒株与７６－１１８等毒株苷酸序     

肾综合征出血热双价纯化疫苗的研制

目的: 研制肾综合征出血热双价纯化疫苗。方法:肾综合征出血热病毒I型LR1 株和II型R22株分别在Vero细胞上接种培养。I、II型病毒培养液依次经β 丙内酯灭活、超滤浓缩、蔗糖区带离心纯化及层析脱糖等处理。将检定合格的I、II型单价病毒原液等量混合后, 用AI(OH)3 佐剂吸附,制备 3批双价纯化疫苗。结果: 该 3批疫苗已经自检和中国药品生物制品检定所复检合格, 并已进行临床试验。结论: 采用Vero细胞培养制备肾综合征出血热双价纯化疫苗方法可行。     

遗传重配获得H5N1流感病毒Vero细胞适应株

目的以传统遗传重配技术选育HSN1流感病毒Veto细胞适应株,制备Vero细胞H5N1流感疫苗。方法以流感病毒Vero细胞适应株A／Yunnan／1／2005Va（H3N2）为母株与反向遗传学技术改造的禽流感病毒疫苗株A／Anhui／1／2005（H5N1）共同感染SPF鸡胚和Vero细胞,用羊抗A／Yunnan／1／2005Va（H3N2）抗体筛选,血抑试验和基因测序鉴定病毒型别,并进行重配株的其他相关生物学试验。结果获得了1株在Vero细胞高产的H5N1流感病毒,重配前后的单价灭活疫苗免疫小鼠抗体血清效价差异无统计学意义（F=0．857,P〉0．05）。结论通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,可以获得H5N1流感病毒Vero细胞适应株。     

流行性乙型脑炎Vero细胞纯化灭活疫苗的研究

目的研制新的流行性乙型脑炎传代细胞疫苗。方法将流行性乙型脑炎弱毒株ＳＡ１４１４２适应于Ｖｅｒｏ细胞,作纯化灭活疫苗,比较不同培养方法病毒在Ｖｅｒｏ细胞的增殖规律,对病毒的浓缩和纯化条件进行研究,并将所制备的疫苗按不同蛋白浓度免疫动物。结果发现普通转瓶培养,可长时间维持病毒的高滴度。在感染病毒后,以无牛血清的培养基替代含牛血清的培养基,不仅可维持病毒的高滴度增殖,而且可多次收获,确立了应用８％ＰＥＧ８０００浓缩病毒液,１５％～６０％的蔗糖密度梯度超速离心纯化的工艺,每剂量为０５μｇ纯化疫苗的中和抗体水平,即可达到与现有商品疫苗相一致的滴度。结论ＳＡ１４１４２株制备的Ｖｅｒｏ细胞纯化疫苗,有望成为一种新的反应轻、效价高的疫苗。     

鼠源性抗汉坦病毒mAb在人体内的动态变化及抗鼠抗体的产生

鼠源性单克隆抗体(ｍＡｂ)为异种蛋白,应用于人体后,半衰期较短,并可能刺激机体产生抗鼠抗体[1]。本文观察了肾综合征出血热(ＨＦＲＳ)患者接受抗汉坦病毒ｍＡｂ治疗后,其血清中鼠ＩｇＧ的代谢及抗鼠ＩｇＧ抗体产生的情况,以为鼠源性ｍＡｂ更好地应用于临床提供理论和实验...     

汉坦病毒山东分离株的分子生物学研究

目的 应用聚合酶链反应(PCR)、核苷酸序列分析等方法，比较山东省家鼠型汉坦病毒的生物学特性。方法 提取汉坦病毒感染细胞的全细胞RNA，逆转录后用聚合酶链反应扩增病毒cDNA。对PCR产物进行核苷酸序列测定。结果 汉坦病毒山东分离株SD25、SD70、SD27和SDl0均为家鼠型汉坦病毒，核苷酸及氨基酸序列与已知SE0型病毒具有较高的同源性，这4株病毒间的同源性高达98％以上。结论 4株病毒分离时间跨度虽有十几年，但相互间变异很小，证实了家鼠型汉坦病毒的稳定性。     

汉坦病毒R36株的基因分型研究

采用汉坦病毒Ｍ片段特异性核苷酸分型引物，对Ｒ３６等７株汉坦病毒进行反转录和聚合酶链式反应扩增鉴定，结果Ｒ３６株仅能被Ⅰ型分型引物扩增出特异性片段，而不能被Ⅱ型引物扩增；ＰＣＲ产物的序列分析结果表明，Ｒ３６与ＨＴＮ型病毒７６－１１８株的同源性为７８．４％，与ＳＥＯ型病毒Ｒ２２株的同源性为６８．１％。文中对Ｒ３６与汉坦病毒其它毒株的核苷酸同源性进行了配对比较。该项研究为从核苷酸水平对Ｒ３６株的分型，提供了科学依据。     

Adaptation of transmissible gastroenteritis virus to growth in non-permissive Vero cells

Summary The CPK cells derived from swine kidney were infected with the attenuated TO-163 strain of transmissible gastroenteritis (TGE) virus, and fused with uninfected Vero cells in the presence of polyethylene glycol. Repeated cocultivation of the fused cells with uninfected Vero cells rendered the virus to grow in Vero cells. The Vero cell-adapted virus acquired the ability to infect and produce cytopathic effects in several other non-permissive cell lines of non-porcine origin. No major differences in viral polypeptides were shown between the Vero cell-adapted TO-163 strain and its parent strain by indirect immunofluorescence and Western blotting using monoclonal and polyclonal antibodies to TGE virus.     

Use of monoclonal antibodies for differentiating strains of the virus causing the hemorrhagic fever with renal syndrome

Using monoclonal antibody and animal immune sera the experiment confirmed the existence of antigenic relationships between the strains of virus of hemorrhagic fever with renal syndrome (HFRS) isolated in the USSR from Clethrionomys glareolus and members of all known serotypes of HFRS virus. Clear-cut differentiation was made from serotypes Apodemus and Rattus, and cross-relationships were shown between the strains isolated in the USSR and Prospect Hill virus (PHV), a member of the serotype Microtus isolated in the USA.     

上海地区肾综合征出血热尸检肾组织病毒检测

目的：研究上海地区肾综合征出血热（ＨＦＲＳ）尸检病例肾脏组织中病毒ＲＮＡ及抗原的分布及定位，方法：用核酸原位分子杂交方法和细胞免疫组化方法，检测了１７例ＨＦＲＳ尸检肾组织中病毒，结果：１７例肾组织中１５例可检测到病毒ＲＮＡ及抗原，病毒ＲＮＡ主要位于肾组织血管壁，毛细血管内皮细胞脉冲有小球毛细血管内皮细胞，病毒抗原主要分布于肾间质管内皮细胞和血管壁中，部分位于肾曲管上皮细胞，以病毒包涵体样颗粒出现。     

肾综合征出血热病毒宫内感染的临床研究

目的 研究肾综合征出血热(HFRS)病毒宫内感染情况及其对婴儿的影响。方法 对妊娠期感染HFRS病毒者，于其分娩时留取母血和脐带血进行抗—HFRS IgG检测，同时对顺产新生儿采取静脉血进行抗—HFRS IgM检测，并应用血凝抑制试验(HI)对HFRS病毒进行分型，另外对顺产的新生儿进行全面查体和定期随访观察。结果 母血抗—HFRS IgG均阳性，死胎的27例脐带血抗—HFRS IgG阳性23例，孕妇痊愈后自然分娩的12例中脐带血和静脉血有2例抗—HFRS IgG阳性，而抗—HFRS IgM阴性。并发现14例顺产新生儿生长发育全部正常。结论 HFRS病毒存在宫内感染，并易致死胎，但对顺产婴儿未发现致畸作用。     

The entry of African swine fever virus into Vero cells

The entry of African swine fever virus into Vero cells has been investigated by both biochemical and morphological techniques. A quantitative electron microscopy analysis of the early steps of the infection has shown that African swine fever virus enters Vero cells by a receptor-mediated endocytosis mechanism. The internalization of virus particles is a temperature- and energy-dependent process, since it did not take place at 4 degrees or in the presence of NaF and 2,4-dinitrophenol. To determine the involvement of acidic intracellular vacuoles in the virus entry pathway we have tested the effect of lysosomotropic agents in the infection. Chloroquine, dansylcadaverine, amantadine, methylamine, and ammonium chloride inhibited African swine fever virus production in Vero cells. Dansylcadaverine and chloroquine did not inhibit virus adsorption and internalization; however, in the presence of these drugs, virus particles were retained in cytoplasmic vacuoles and early viral RNA and protein synthesis were not detected, indicating that these compounds inhibit an early step in the infectious cycle, probably the uncoating of the virus particle.     

Virus-specific proteins and RNA of the virus of hemorrhagic fever with renal syndrome

Virus-specific proteins G1, G2, and N with molecular weights of 70, 55-57, and 50 kilodaltons, respectively, were detected by radioimmunodiffusion tests in VERO E-6 cells infected with strains of virus of hemorrhagic fever with renal syndrome (HFRS) isolated in the European USSR from a patient with HFRS, a fatal human case of HFRS (the strains K-27 and P-360) and from a bank vole (strain CG-1820). The sera from human convalescents after HFRS in the European USSR and rat sera prepared with the CG-1820 strain precipitated proteins possessing similar electrophoretic characteristics from a lysate of cells infected with the CG-1820, K-27 and P-360 strains. The sera from human HFRS convalescents in the Far East did not precipitate protein Gl. The viral RNA derived by immunosorption method from intracellular nucleocapsids of CG-1820 strain and strain 4590 (isolated from Ap. Peninsulae in the Far East) contained 3 classes of molecules: L, M, and S. L- and M-RNA of these strains had the same molecular weight (1.62 and 1.38 megadaltons). The molecular weight of S-RNA of the strain 4590 was 0.76 megadalton and that of the CG-1820 strain 0.83 megadalton. It is assumed that there are species differences among the viruses, causative agents of HFRS, circulating in the European and Far East regions of the USSR.