甲型流行性感冒病毒自然温度敏感株基因损害定位

采用重组试验和聚丙烯酰胺凝胶电泳(PAGE)技术,把晚期甲3型流感病毒自然ts突变株齐防79-39的ts损害定位在膜蛋白(M)基因上。但互补试验表明,齐防79-39与M基因损害的WSN标准株ts51可以发生互补,这是基因内互补的一个证据。PAGE技术证实,新甲1型流感病毒自然ts株津防77-78的M基因上确有损害。互补试验证明齐防79-39属于一个互补组,而津防77-78与ts51同属于另一个互补组。 本文结果还表明,晚期甲3型齐防79-39的ts损害基因可能是由甲3型野毒株自发突变所产生,而并非通过在自然界与新甲1型重组而获得。     

甲3型流行性感冒病毒血凝素的pH特征

本文报告,pH9.6碳酸缓冲液对甲3型流感病毒的血凝滴度有明显降低作用,对甲1型和乙型仅有轻微影响,对甲2型的影响则介于两者之间。用不同pH的碳酸缓冲液、磷酸缓冲液及盐水等,测定甲3型流感病毒的血凝滴度,结果表明高pH对其有明显影响。分别具有甲3、甲2或甲1血凝素的重组株,在pH9.6碳酸缓冲液中,其血凝素的稳定性也和上述结果一样,即具有甲3血凝素的重组株,其血凝素对pH9.6碳酸缓冲液最敏感;甲1重组株的血凝素较稳定:而具有甲2血凝素的重组株则介于两者之间。利用此pH特征测定新分离的经血凝抑制试验鉴定为甲3型和乙型流感病毒,得到同样结果,因此有可能利用此pH特征对新分离的甲3型流感病毒进行初步鉴定。     

流感病毒表面抗原血凝素基因在枯草杆菌中的克隆和表达

前言 流行性感冒仍然是全世界最严重的流行病之一。其病原主要是甲型流感病毒,分为甲_1、甲_2、甲_3三个亚型。当前流行的是甲_1和甲_3型。能有效预防流感的措施是用疫苗免疫。血凝素(HA)是流感病毒表面最重要的抗原,能诱导产生流感病毒中和抗体。从纯化的流感病毒颗粒中提取的血凝素,其免疫性与灭活的流感疫苗相似,但作为亚单位疫苗使用,因生产成本过高,并未广泛推广。     

四价流感病毒裂解疫苗B型血凝素含量的检测方法

目的建立能准确测定四价流感病毒裂解疫苗中B型血凝素含量(单向免疫扩散法,SRD)的检测方法。方法采用双价抗原参考品SRD法对四价流感病毒裂解疫苗中两种B型血凝素含量进行测定。将B1与B2抗原参考品按质量浓度1∶1混合制备为双价抗原参考品,双价抗原和待检样品与10%裂解剂按9∶1比例裂解30 min,分别加入到含有抗血清参考品的1.5%琼脂糖凝胶板上,每孔10μL,置20~25℃放置18~24 h,经干燥、染色、脱色,测量结果。验证双价抗原SRD法的重复性和准确性。结果双价抗原SRD法测定的血凝素含量平均值比单价抗原SRD法更接近理论配制值,故双价抗原SRD法比单价抗原SRD法能更能准确检测QIV中两种B型血凝素含量,经验证双价抗原SRD法的重复性及准确性良好。结论双价抗原SRD法提高了四价流感病毒裂解疫苗B型血凝素含量检测的准确性,为精确测定四价流感病毒裂解疫苗中B型血凝素含量提供了有效的方法和数据支持。     

几株甲型流感病毒神经氨酸酶(NA)理化特性和抗原性的比较

以两株甲2型流感病毒株贵57-1、甘66-3(H2N2)和两株甲3型流感病毒株京科68-1、穗防74-315(H3N2)为代表株,测定温度、pH及金属离子对感染的鸡胚尿囊液病毒神经氨酸酶(NA)活性的影响,并用NA交互抑制试验检查贵57-1NA与穗防74-135NA之间的N抗原变异情况。 结果表明,在50℃加热30分钟4株病毒的NA活性仅下降原活性的10％以下;但56°C30分钟加热,两株甲2型病毒(H2N2)的NA活性明显下降,仅为原活性的12％以下;而两株甲3型(H3N2)则分别为原活性的34％及37％。根据Paniker和Tams等的报道,N2的活性对45℃及56℃加温比N1更稳定。似乎表明,变异着的NA抗原有越来越耐热的趋势。     

单克隆抗体对感染流行性感冒病毒的小鼠的疗效观察

治疗流行性感冒(简称流感)对年老体弱者有着重要意义。由于流感病毒的一个新亚型出现后,往往要经历十几年至20多年的流行,因此选出具有亚型内共同抗原决定簇的McAb,用于治疗流感是可行的,文献也曾有过报道。前文已报道关于甲3型流感病毒McAb的建立,本文用抗甲3型流感病毒上海/32/84血凝素McAb,与1977年以来分离的甲3型流感     

猪与人甲_3型流感病毒的比较研究——Ⅰ、抗原性及多肽结构分析

比较1978、81、82年从猪中分离出的甲_3流感病毒与同年代人甲_3流感病毒的抗原性及多肽结耕。从血清学、血凝素肽图以及SDS-PAGE电泳图都表明逐年猪甲_3与人甲_3血凝素(HA)及神经氨酸酶(NA)抗原性密切相关。而且膜蛋白与核蛋白也基本相同。更明确表明人的甲_3流感病毒年年不断地极易传给猪。猪中有了甲_3病毒株其NP迁移位置与其它毒株略有不同,文中对此进行了讨论。用~(125)I标记血凝素肽图比较1979、1981、1982、1983年猪与人甲_3毒株,逐年在肽图上都有明显差异,同年不同时间分离的毒株之间,肽图上也略有改变,更进一步表明此法的灵敏性。     

用单克隆抗体选择具有潜在流行病学意义的A/曼谷79—1流感病毒变异株

用甲3型流感病毒A/曼谷79-1血凝素特异的单克隆抗体,对我国从1979—1984年分离的45株H3N2病毒进行了抗原分析,并与用单克隆抗体选择的51株A/曼谷79-1变异株作了比较。发现后者同1979年以来我国流行的H3N2流感主流株高度相似。这证明,用单克隆抗体可选出有流行潜力的变异株。本文就通过单克隆抗体选择、提前获得甲型流感亚型内变种的可能性进行了讨论。     

基于血凝素关键序列的流感病毒疫苗

流感病毒的血凝素(HA)位于流感病毒包膜上,在流感病毒吸附和穿入宿主细胞的过程中起着重要作用.血凝素以三聚体的形式镶嵌在病毒包膜上,每个单体糖蛋白是由两个经二硫键连接的蛋白亚单位组成,即HA1和HA2.血凝素属Ⅰ型膜蛋白,其一级结构含有4个结构域:信号肽(前导序列)、胞浆域、跨膜域和胞外域. 血凝素是流感病毒最主要的表面抗原,它能够诱导机体产生相应的中和抗体以中和病毒.血凝素一般含有5 个抗原决定簇,流感病毒的流行与其抗原结构的变化密切相关.主要就血凝素的结构和功能、流感病毒疫苗以及以血凝素基因关键序列为基础的流感病毒疫苗进行阐述.     

抗甲3型(H3N2)流行性感冒病毒单克隆抗体的建立

将SP2/0 Ag小鼠骨髓瘤细胞与经蔗糖梯度离心纯化的甲3型流感病毒沪防/32/84(H3N2)免疫的BALB/C鼠脾细胞融合,经初步克隆获得了25个能稳定分泌抗甲3型流感病毒单克隆抗体(McAb)的杂交瘤细胞株,部份McAb的一些生物学性状如下。 经ELISA测定25个腹水McAb、除4株为10~(-4)外,其余滴度在10~(-5)～10~(-(?))间。此25个McAb中,16个为抗血凝素McAb,其中15个的血凝抑制效价在1:1,280～1:20,480,1个为1:320,在鸡胚内均有不同     

The highly conserved arginine residues at positions 76 through 78 of influenza A virus matrix protein M1 play an important role in viral replication by affecting the intracellular localization of M1

Influenza A virus matrix protein (M1) plays an important role in virus assembly and budding. Besides a well-characterized basic amino acid-rich nuclear localization signal region at positions 101 to 105, M1 contains another basic amino acid stretch at positions 76-78 that is highly conserved among influenza A and B viruses, suggesting the importance of this stretch. To understand the role of these residues in virus replication, we mutated them to either lysine (K), alanine (A), or aspartic acid (D). We could generate viruses possessing either single or combination substitutions with K or single substitution with A at any of these positions, but not those with double substitutions with A or a single substitution with D. Viruses with the single substitution with A exhibited slower growth and had lower nucleoprotein/M1 quantitative ratio in virions compared to the wild-type virus. In cells infected with a virus possessing the single substitution with A at position 77 or 78 (R77A or R78A, respectively), the mutated M1 localized in patches at the cell periphery where nucleoprotein and hemagglutinin colocalized more often than the wild-type did. Transmission electron microscopy showed that virus possessing M1 R77A or R78A, but not the wild-type virus, was present in vesicular structures, indicating a defect in virus assembly and/or budding. The M1 mutations that did not support virus generation exhibited an aberrant M1 intracellular localization and affected protein incorporation into virus-like particles. These results indicate that the basic amino acid stretch of M1 plays a critical role in influenza virus replication.     

Differentiation Between Herpes Simplex Virus Type 1 and Type 2 Strains by Immunoelectroosmophoresis

A method has been elaborated to differentiate between herpes simplex type 1 and type 2 viruses by immunoelectroosmophoresis. With rabbit immune sera cross-absorbed with heterologous virus antigen, a distinct difference was shown between the two virus types. Herpes simplex type 1 virus tested against cross-absorbed type 1 antiserum gave two precipitin lines. Herpes simplex type 2 virus gave one precipitin line when tested against cross-absorbed homologous serum. When the viral antigens were tested against cross-absorbed heterologous immune sera, no or only very weak precipitin reactions were observed. The test is easy and rapid, requires relatively small quantities of antigen and antibody, and is suitable for typing of herpes simplex virus in diagnostic routine work.     

Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells

L cells expressing either the A/NT/60/68 nucleoprotein or the A/PR/8/34 (H1) hemagglutinin by DNA mediated gene transfer were used to investigate recognition by influenza A specific cytotoxic T lymphocytes (CTL). A subpopulation of CTL that recognized the H1 hemagglutinin was detected in mice primed with either A/PR/8/34 (H1N1) or A/JAP/305/57 (H2N2) influenza viruses. However, neither CTL from mice primed with A/NT/60/68 (H3N2) nor the recombinant virus X31 (H3N2) showed any activity on L cells expressing H1. These results showed that the majority of fully crossreactive CTL do not recognize the hemagglutinin molecule. A comparison between nucleoprotein and hemagglutinin transfected L cells reveals the nucleoprotein as the major target for CTL that are crossreactive on the three pandemic strains of human influenza A virus.     

Antigen mimicry involving measles virus hemagglutinin and human respiratory syncytial virus nucleoprotein.

Intergenic antigenic relationships between measles virus and respiratory syncytial (RS) virus-specific structural components were studied by using monoclonal antibodies. Of 75 monoclonal antibodies against these components, only one, an anti-measles virus hemagglutinin monoclonal antibody, cross-reacted. Immunofluorescence analysis of measles virus- and RS virus-infected cells with this monoclonal antibody showed qualitatively different staining patterns which indicated that the antigen involved in cross-reaction was the RS virus nucleoprotein or phosphoprotein. A radioimmunoprecipitation assay showed the antigen to be the nucleoprotein.     

Immunological and genomic analyses of two serotypes of avian paramyxovirus isolated from wild ducks in Japan

A total of 11 avian paramyxoviruses isolated from migrating feral ducks in Niigata, Japan, were characterized by serological and genomic analyses. Hemagglutination inhibition and immuno-double-diffusion tests with antisera specific for the isolated hemagglutinin-neuraminidase polypeptides of reference strains indicated that, of these, eight isolates possessed hemagglutinin-neuraminidase antigen closely related to that of duck/Hong Kong/D3/75, and the remaining three isolates possessed antigen closely related to that of duck/Hong Kong/199/77. RNA analysis of the eight isolates identified serologically as duck/Hong Kong/D3/75 by oligonucleotide mapping revealed that these isolates were genetically very similar to each other but different from the reference strain and isolates reported previously. The oligonucleotide maps of duck/Hong Kong/199/77-like isolates appeared to be very similar to each other, suggesting the same origin, but not to the duck/Hong Kong/199/77 virus.     

Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.     

Sequential mutations in the NS genes of influenza virus field strains.

The complete nucleotide sequences of the NS genes from three human influenza viruses, A/FM/1/47 (H1N1), A/FW/1/50 (H1N1), and A/USSR/90/77 (H1N1), were determined. Only five single-base differences were found within the sequences of the A/FW/1/50 and A/USSR/90/77 NS genes, thus confirming earlier data suggesting that the 1977 H1N1 viruses are closely related to virus strains that were circulating around 1950. Comparison of all three sequences with those from A/PR/8/34 and A/Udorn/72 viruses illustrates that these genes (with the exception of that of the A/USSR/90/77 strain) evolve through cumulative base changes along a single common lineage. A nucleotide sequence variation of approximately 2.2 to 3.4% per 10 years was determined for the NS gene segments. Extensive size variation was also observed among the NS1 proteins of the various human viruses. The A/FM/1/47 NS1 protein, which consists of 202 amino acids, is 15% shorter than the A/Udorn/72 NS1 protein, which consists of 237 amino acids.     

Isolation, sequencing and phylogenetic analysis of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 and H3N8

We report here on the isolation and sequencing of the hemagglutinin, neuraminidase and nucleoprotein genes of the Chilean equine influenza virus subtypes H7N7 (A/equi-1/Santiago/77, Sa77) and H3N8 (A/equi-2/Santiago/85, Sa85). The sequences obtained allowed a variability analysis, which indicated significant differences when compared with other isolates. We found that Chilean isolates are more similar to the North American variety than to European isolates. Isolate Sa77 is a good candidate for inclusion in a vaccine as it is the latest isolate of the subtype H7N7 and is probably better-adapted to the equine host. Isolate Sa85, of subtype H3N8, also appears to be a good candidate since it has no significant differences in the main antigenic sites with recent isolates.     

Group-specific and type-specific gel diffusion precipitin tests for bluetongue virus serotype 20 and related viruses

Using antigens prepared from cell cultures infected by bluetongue (BLU) virus type 20 (BLU-20), and sera from cattle which had recovered from experimental infection by that virus, two distinct precipitin reactions were demonstrated by immunodiffusion. Two distinct gel diffusion precipitin tests were developed based on these reactions. The antigen of one was common to BLU-20 and two other Australian BLU isolates, CSIRO 154 (BLU-21) and CSIRO 156 (BLU-1). It was therefore concluded to be a group-specific test. The antigen of the second appeared to be unique to BLU-20. The test based on this antigen correlated well with the virus neutralization test for BLU-20 and it was therefore concluded to be type-specific. Similar methods applied to a virus of the Palyam (PAL) group demonstrated two precipitin reactions of similar broad (group) and narrow (type) specificity.