流行性出血热病毒在Vero一E6细胞内引起细胞病变作用的新发现

自McCormick等发现Vero-E6细胞对流行性出血热病毒的敏感性后,国内外已有不少学者应用该细胞进行病毒分离和试验研究,但均未发现病毒在该细胞内规律性病变。病毒的存在和滴度只能按免疫荧光法(IFA)证实和测出。本文应用我国分离的出血热病毒在Vero-E6细胞上培养和观察,发现病毒在该细胞上出现有规律的病变并能测出病毒滴度,病变能被特异性中和抗体中和。现将结果报道如下。     

流行性出血热病毒的蚀斑形成技术

国外已有报道,流行性出血热病毒(EHFV)能在Vero-E6细胞上形成蚀斑。由于蚀斑试验和蚀斑减少试验用于测定病毒滴度及中和抗体效价较其它方法准确,且特异性高,适用于测定出血热病毒间抗原性差异,感染或免疫后中和抗体水平。但由于EHFV在细胞内繁殖培养时间较长,蚀斑形成的细胞培养条件要求较高,目前国内尚未见成功的报道。我们基本     

应用微量细胞病变中和试验法(MCPENT)对我国EHF病毒株和病人血清进行血清分型

本文应用微量细胞病变中和试验(MCPENT)对我国22株不同来源的流行性出血热病毒(EHFV)进行血清分型研究结果除一株具有广谱抗原性外,所有毒株均可准确地被分为血清1型和血清2型,其分型结果与用空斑减少中和试验(PRNT)完全一致。此外,MCPENT法还能对不同流行区EHF病人血清进行分型诊断。此法简便、经济、准确可靠,便于推广应用。     

流行性乙型脑炎病毒血清抗体半微量空斑抑制法的改进

本文报道一种检测流行性乙型脑炎(简称乙脑)中和抗体的简化空斑抑制试验方法,用BHK21传代细胞在24孔塑料板上进行试验,在细胞未形成单层时,将病毒直接接种在刚消化的细胞悬液内,孵育后加甲基纤维素复盖物,再培养,细胞层经染色后即可计算空斑数。 用该法检查47名儿童乙脑疫苗免疫前后的中和抗体,与常规的鸡胚细胞全量法或BHK21传代细胞微量中和试验法作了比较,结果表明本法与其它常规法的敏感性一致,且具有简便快速、节省人力和原材料、空斑形成率高的优点,便于大量血清学检测。     

SARS-CoV细胞空斑试验

目的建立适用于生物安全三级实验室操作的SARS．CoV空斑试验方法。方法SARS－CoVl0倍系列稀释接种Vero－E6细胞,用0．6％琼脂糖覆盖中性红着色（琼脂糖．中性红法）或覆盖1％甲基纤维素,4％甲醛固定,结晶紫染色（甲基纤维素－结晶紫法）,空斑计数。结果琼脂糖－中性红法病毒滴度为6．14Lg pfu／mL,甲基纤维素－结晶紫法病毒滴度为6．57Lg pfu／mL。结论二种空斑试验方法相比,病毒滴度无明显差异。甲基纤维素－结晶紫空斑试验法形成的空斑比琼脂糖－中性红法清晰、操作简单安全、结果可长期保存。     

应用型特异性血清对肾综合征出血热毒株进行空斑减少中和试验分型

本文应用特异性出血热抗血清对国内外１４株出血热病毒进行空斑减少中和试验分型,结果不同毒株对两型持异性血清的中和效价大多相差８－１６倍,可以明显确定为出血热血清Ⅰ型或Ⅱ型。该方法用以出血热新分离株的分型不必制备分离株免疫血清,使鉴定更简便,并缩短时间。     

长爪沙鼠腹腔巨噬细胞对流行性出血热病毒的敏感性

本文就长爪沙鼠腹腔巨噬细胞(MΦ)对流行性出血热病毒(EHFV)的敏感性进行了试验,用3株野鼠型(A9、R3、76-118)和1株家鼠型EHFV(R22),并用对EHFV敏感的Vero-E6细胞作对照,结果沙鼠腹腔巨噬细胞感染EHFV后,第1代第8天前即可查见明显的特异性荧光,第16天左右达高峰,病毒滴度≥10~(-7.5),与Vero-E6细胞比较,培养上清液的病毒滴度,沙鼠MΦ较Vero-E6细胞高1～4个对数,当感染病毒量很低时,在Vero-E6细胞内测不出特异性抗原,而在沙鼠MΦ内病毒仍可繁殖,滴度达10~(-5.(?)),中和试验、间接免疫荧光检测和荧光阻断试验均证明,沙鼠MΦ内繁殖的病毒确实为EHFV。     

出血热病毒株的分型和抗原性比较的进一步研究

本工作进一步对分离自不同来源的14株出血热病毒用空斑减少中和试验(PRNT)方法进行了分型。以不同株的家兔免疫血清对国际上血清Ⅰ型和Ⅱ型参考毒株进行试验,除分离自褐家鼠的K_(24)-株外,其余13株均可定为血清Ⅰ型或血清Ⅱ型用已知的Ⅰ型和Ⅱ型出血热高价免疫血清与K_(24)株进行中和试验,根据两型免疫血清的中和效价则可将K_(24)株定为血清Ⅱ型,但是K_(24)株免疫血清对6株Ⅰ型病毒和4株Ⅱ型病毒的中和效价几乎相同(相差≤2倍),表明K_(24)株是一株具有广谱中和抗原的出血热病毒。     

速成单层法半微量病毒空斑技术的研究

本文报道一种不需预先制备细胞单层的速成单层法半微量病毒空斑技术(简称速成法)及其在空斑中和试验与空斑纯化试验中的应用。作者以脊髓灰质炎病毒为代表,对速成法的实验条件、可靠性、敏感性、重复性等作了系统研究,并以此法对单纯疱疹病毒、呼吸道合胞病毒和水泡性口炎病毒进行了空斑滴定。结果提示速成法简单快速、经济方便、敏感稳定、可靠易行,可能具有较大的实用和推广价值。     

流行性出血热免疫球蛋白的研制

本文首次报告采用纯化灭活流行性出血热（ＥＨＦ）Ⅰ型疫苗免疫健康献血员,采集ＥＨＦ抗体高滴度的血浆,用低温乙醇法及盐析法分离纯化三批ＥＨＦ免疫球蛋白。结果表明：（１）采用０,１,３,４（月）或０,１,４,５（月）免疫程序,疫苗剂量１～２ｍｌ（０．１５～０．３０ｍｇ蛋白）,受免献血员血清平均抗体滴度可达１∶４０６（ＥＬＩＳＡ）或１∶１１２（ＲＰＨＩ）。（２）通过生化检定,三批制品的电泳纯度为９７．０５％,９６．８４％,９９．２６％;ＩｇＧ单体和二聚体含量为８９．５５％,９１．３０％和９８．２１％。（３）用空斑抑制中和试验及免疫印染试验证明所纯化的免疫球蛋白具有抗ＥＨＦ病毒特异性。（４）三批ＥＨＦ免疫球蛋白的效价测定,结果为ＥＬＩＳＡ滴度≥１∶５１２,ＲＰＨＩ滴度≥１∶１０２４,ＰＲＮＴ（中和抗体）滴度１∶４０。按１６％蛋白计,ＥＨＦ免疫球蛋白的效价可达原料血浆的１０倍以上。（５）无菌、安全、毒性及热原质试验检定结果,全部通过《中国生物制品规程》要求。     

流行性出血热(EHF)病毒半微量甲基纤维素空斑法的建立

本文报道EHF病毒一种新的空斑形成按术,即半微量甲基纤维素空斑法的建立及其应用。6株来源不同的毒株均可在V_(ero)-E_6细胞或V_(ero)细胞上形成清晰的空斑,形成的空斑能被特异抗EHF病毒血清所中和。其敏感性与用琼脂糖作覆盖物的空斑法一致。该法具有操作简便、快速等优点,适用于EHF的病原学和血清学研究。     

汉坦病毒沟3株PCR分型

汉坦病毒 (HV)沟 3株在以往报道的免疫学中和效价检测 ,为一株中和抗原广谱的毒株。本研究对这一毒株经过两次挑斑纯化 ,并经PCR方法进行分型检定 ,初步认为沟 3毒株为SEO型HV病毒。     

斑点杂交生物素法检测流行性出血热病毒RNA

为寻找一种用于检测流行性出血热病毒的分子杂交方法,以生物素-7-dATP标记流行性出血热病毒(EHFV)R_(22)株M片段的cDNAR_3克隆作探针,与人源性的EHFVH-114、H-435株RNA基因组进行斑点杂交,得到阳性结果,可检出5pg的cDNA或RNA。此探针与疱疹病毒DNA不出现杂交信号。以上结果说明这种标记探针具有EHFV特异性,可以扩大应用范围,结果还表明动物源性和人源性EHFV均具有共同的保守核苷酸序列。     

Accuracy and repeatability of a micro plaque reduction neutralization test for vaccinia antibodies.

The detection of neutralizing antibodies against vaccinia virus is a valuable tool for the investigation of previous smallpox vaccination. Compulsory smallpox vaccination ended in Brazil during the early 1970s, although the vaccine was available until the late 1970s. The threat of smallpox as a biological weapon has called the attention of public health authorities to the need for an evaluation of the immune status of the population. Based on our previous experience with a micro plaque reduction neutralization test (PRNT) for the evaluation of yellow fever immunity, a similar test was developed for the detection and quantification of vaccinia neutralizing antibodies. A cross-sectional study to test the repeatability and validity of plaque reduction neutralization test (PRNT) for vaccinia antibodies was performed in 182 subjects divided into two categories: subjects above 31 years old and the other > or = 35 years old. Cases were subjects considered to have been vaccinated with vaccinia virus if they declared vaccination history or evidenced vaccination marks. The assay is carried out in 96-well plates, provides results within 30 h, is easily performed, has good sensitivity (92.7%) and specificity (90.8), excellent repeatability (ICC 0.89 (0.88; 0.92)) and is thus suitable for use in mass screening of a population's antibody levels.     

A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.