The promolion of membrzme fusion by the fusion (F) protein of human parathfluenza vires 3 (fiPIV3) is dependenl on a vies specific eontnbution from the fiemagglulinin-neuraminidase (HN) protein By evaluarion of chimeric hPIV3-Newcasfle disease vires (NDV) HN proteins, we have previously show n that hPl V3 F-speci ficity is determined by a domain that extends lYnm the middle of the membrane anchor to the 82rid residue in the ectodomain [Virology 209. (1995) 457; Arch Virol 13 (1997) 115]. If the corresponding NDV-derived residues replace the two C-terminat residues in this domain, no fusion is dcteeled. However. these substitutions restore a glycosyladon sile present in NDV HN, but not in hPIV3 HN. Deletion of this site from a nested set of chbneras with fiPIV3-derived N terminal porlions of decreasing length partially restores fusion, suggesting that an ofigosacchande near the top of hPIV3 HN stalk modulates fusion In addition, further mutational analyses show that a chimera with only 125 N temdnal hPIV3-dedved residues (72 in the stalk) actually promotes fusion more efficiently than Ihe wt protein These findings Iocalize the C ternrinus of the F-specific domain in hPlV3 HN n full 10 residues closer to the membrane than previously shown

Objective: To investigate the correlagon between rubella Jrus (RuV) antigen in peripheral Jymphocytes, the im mLine status and RuV infection in the central nervous system (CNS). Metbods: BALB/c mice were used as a model and treated with immunoaffecting medicines. Then, the mice were infected with RuV via the abdominal cavity, and the antigen lever in peripheral lymphocytes was examined 1.3, 7 and 14 days postinfection. RuV in the CNS was detected by immunohistocbemical meth ods. BALB/c mice were given dexamethasone and cytoxan before infection with the RuV JR23 strain. Immune functions and RuV invasion of the CNS were assayed on day 21 postinfection via the abdominal cavity, and their relationship was analyzed. Results: The mean antigen

The ectodomain of the paramyxovirus haemagglutinin neuraminidase (HN) glycoprotein spike can be divided into two regions: a membrane-proximalp stalk-like structure and a terminal globular domain. The latter contains all the antibody recognition sites of the protein, as well as its receptor recognition and neuraminidase (NA) active sites. These two activities of the protein can be separated by monoclonal antibody functional inhibition studies and mutations in the 91obular domain. Herein, we show that mutation of several conserved residues in the stalk of the Newcastle disease virus HN protein markedly decrease its NA activity without a significant effect on receptor recognition. Thus, mutations in the stalk, distant from the NA active site in the globular domain, can also separate attachment and NA. These results add to an increasin9 body of evidence that the NA activity of this protein is dependent on an intact stalk structure.

To construct an expression vector containing the E1 glycoprotein gene of rubella virus for the study on the effect of mutation of the E1 gene glyeoprotein and the analysis of phylogenetic differences of sequences,the gene encoding the E1 envelope glcoprotein was amlified from rubella virus, Jinan strain JR23, by RT-PCR and ligated into PMD-18T vector. The clones that camied the E1 gene were identified after amp selection and analysis of restrition enzyme digenstion. After sequencing this gene was analyzed by Danstar and Winstar prograns, and the map of phyolgenetic tree was dramw. The colone of E1 glycoprotein was thus constructed. It was found that the sequence differeces between JR23 strain and the TCRB strain from Japan and those between JR23 strain and Thomas strain of England were rather small with difference values of 0.9% and 1.2% respectively. Yet those between JR23 strain and BRD2 strain from Beijing and those between JR23 strain and XC379 strain from Hong Kong were comperatively larger with difference values of 7.6% and 7.3% resoectivesly. The sequece of JR23 strain with other strains was less than 3% except the NC strain (3.7%). It concludes that the construction of E1 glcoprotein gene offers an approach to study the relationship between structures and functions of E1 gene and its gene products. In the phlogenetic tree, it shows that there are significant differences in the sequences of rebella vinus isolated in China, and this might be helpful to develop an effective vaccine.

目的 找到副牯病毒融合蛋白（F）分子上与血凝素-神经氪酸酶（HN）相互作用的特异性区域，弄清F融合细胞的分子机理。方法 采用基因定点突变法，创造一个酶切位点，得副突变株，然后用基因重组的方法得到各种重组株。并于真棱细胞内进行表达。Gimsa染色和指示基因法检测细胞融合功能，荧光强度分析（FACS）检测表达效率情况。结果 在将各有关F基因片段进行交换之前，创造一个酶切位点时所得突变株的细胞融合功能与野毒株相同；将新城疰病毒（NDV）F和副流感病毒（PIV）F在膜穿人部分和躯干部分交接处交换时。不影响细胞融合功能；进一步将F2进行交换时，也不影响细胞融台功能；而将F的头部进行交换时。则检测不到细胞融合作用，FACS分析表明，F没有达到细胞表面，结论 F分子上与HN相互作用的特异性区域位于膜穿大部分和躯干部分交接处与F1和F2交接处之间，即F1除去膜穿人部分和足部的剩余部分。

为了研究糖化作用对新城疫病毒（NDV）HN糖蛋白生物学活性的影响，特别是对HN保细胞融合作用的影响，采用基因定点突变技术分别去掉HN分子上的4个糖化位点，然后检测各突变株的细胞表而表达情况、受体识别特性、神经氨酸酶活性、促细胞融合作用、免病沉淀特性等。结果表明，将野毒株NDV HN的细胞表面表达效率定为100%时，K198R-HN突变株的表达效率为82.6%；而对二者的G1、G2、G3和G4 4个糖化位点分别进行定点突发时，得到8种突变株。它们的表达效率均有不同程度的降低，D198R-HN-G2和D198R-HN-G4两种突变47.95%、68.9%、42.67%和41.10%；而D198R-HN突变株的G1、G3和G4突变株的受体识别活性突变前后变化不明显，只有D198R-HN-G2突变株的受体识别活性得以恢复较多，从原来的10.96%恢复到32.88%。野毒株HN突变后神经氨酸酶活性普遍降低，尤以G4影响明显，公为野毒株的9.60%而D198R-HN突变株突变后神经氨酸酶活性普遍升高，尤以G2恢复最高，由原来的0.45%恢复到7.59%，野毒株HN的G1、G2、G3和G4突变前后细胞融合情况变化不大；而D198R-HN的G1、G2、G3和G4P突变后，D198R-HN-G1、D198R-HN-G3、D198R-HN-G4没有变化，但D198R-HN-G2使D198R-HN的细胞融合活性得以恢复30.90%。野毒株HN电泳时吴现1条较宽的泳带，当突变掉1个糖化位点时，泳动速度加快。D198R-HN突变株及D198R-HN-G1、D198R-HN-G3和D198R-HN-G4 HN突变株电泳时，呈现两条模糊不清的条带。但D198R-HN-G2突变株HN电泳时，其条带变得窄而锐利，且泳动速度快。上述结果说明糖链能影响HN的表达或从细胞浆运输到细胞表面，G2对HN的受体识别活性影响较大，推测G2糖链部分结构的改变影响到了HN G2周围的表型特性，从而导致神经氨酸酶活性，促细胞融合作用的改变。

为了探讨风疹病毒（rubella virus, RV）包膜粮蛋白E2的基因与蛋白的遗传与变异特点，利用DNAstar软件比较JR23株与GenBank中各参考株E2的基因与多肽序列，分析不同RV株E2基因之间的同源性及E2蛋白功能关键区的氨酸变异。结果表明，RV E2基因序列比较保守，但JR23析E2基因序列与其它9个分离析之间有明显差异，变异主要集中在484nt-554nt。同源性为90.3%-92.6%之间，与美国疫苗株RA27/3同源性最高，与美国疫苗株HPV77同源性最低，GenBank中的参考株间同源性较高，在96.3%-100.0%之间。E2多肽变异集中于161aa-185aa，但在E2功能关键区，如N端序列、N联粮基化位点、胞质区等则无明显变异。表明RV E2基因序列相对保守，虽然JR23株与GenBank中各参考株的E2基因序列有明显差异，但JR23株E2蛋白在功能关键区表现出遗传稳定性。此结果为阐明RV分子流行病学特征提供了理论依据，也为研究RV分子进行特征病毒与宿主细胞的相互作用奠写了坚实的基础。

为了确定副粘病毒融合蛋白（F）分子上活性位点中亮氨酸在F的细胞融合作用中的作用。弄清F融合细胞的分子机理，采用基因定点突变法创造一个酶切位点，用酶切反应初步筛选突变株，然后用DNA序例分析进一步确定，并在真核细胞内时行表达，Giemsa染色和指示基因法检测细胞融合功能，荧光强度分析（FACS）表达效率。结果表明，hPIV3F第460位亮氨酸（L）和第474位亮氨酸（1）分别突变成丙氨酸（A）（L460A和147A）时，细胞融合功能分别只相当于野毒株的51.24%和48.73%；而第467位L和第481位L分别突变成A（L1467A和L481A）时，细胞融合功能则无明显改变；当第460和467位L同时突变成A（L460A-L467A）时，细胞融合功能降低了79.13%；而474位1和第481位L同时突变成A（1474A-L481A）时，细胞融合功能无明显改变。NDV F第481位L和488位L分别突奕成A（L481A和L488A）时，细胞融合功能分别降低了75.22%和80.04%；将二者共同突变时，则进一步降低，只有野毒株的10.13%。各突变株的表达效率没有改变。说明F分子上与HN相互作用的L，突变后细胞融合作用不同程度下降。

目的 建立一种定量分析副粘病毒包膜糖蛋白融合细胞功能的方法。方法 将vTF-7重组痘苗病毒感染BHK21细胞后，分别转染侍测基因DNA和pCIN17β-gal DNA，16h后将两个细胞群混合，37℃ 15h后用NP-40裂解细胞。取上清进行显色反应，在SOFTMAX软件支持下用ELISA自动测定性在570mm测吸光度A值。结果 指示基因法与细胞计数法有密系，r=0.9890（P<0.01）；与面积断法的符合率达100。最佳主要应成条件包括：16mmol/L底物浓度；显色反应20～25min；1μgDNA转染量；每种细胞量接种1×105个。结论 指示基因法是一种非常敏感、特异、可重复性好的细胞融合定量分析方法。可用于副粘病毒细胞融台作用功能的研究。并可作为其他病毒细胞融合作用研究的参考，具有较大的推广应价值。

利用超薄切片电子显微镜技术对本室分离的风彦病病毒（Rv）JR23株在BHK21细胞中的形态及形态发生过程进行了研究。同时与Rv标准野毒株Gos-10作了比较。结果表明，Rv JR23株盛染BHK21细胞后6h开始干细融浆内观察刊病毒鞭粒，96h达到高峰。病毒颗粒呈圆形。有双层脂质包膜包绕，直径45～75nm。核衣壳25～35nm。细胞裴内见到大量病毒相关颗粒，直径20～30nm病毒包膜米白于细胞禁中的空泡膜或细胞膜。被RV感染的细胞袋中还观察到“繁殖复合体”，由膜性结构包绕着许多类似病毒颗粒的囊泡构成。两株RV在形态与形态发生方面未发现差异。