苹果茎沟病毒的提纯和检测

用生物学和血清学方法从苹果分离鉴定出苹果茎沟病毒G-2和TC-3分离物,采用皂土澄清、15％蔗糖垫超离心、10％～40％蔗糖梯度离心等步骤,获得提纯的病毒样品,紫外吸收比值A_(260/280)为1.15。13％SDS-PAGE法测定的病毒外壳蛋白分子量为31000。用提纯的苹果茎沟病毒G-2分离物制备的兔抗血清,PTA-ELISA测定的效价为1/64000,特异性强。用PAS-ELISA可成功地检测出苹果茎沟病毒,此外还试验了DAS-ELISA,并证明其检测苹果茎沟病毒的效果与PAS-ELISA一致。     

Serological relationship and detection of bean common and bean yellow mosaic viruses in agar gel

Microprecipitin tests demonstrated a distant serological relationship between intact virus particles of BCMV and BYMV. Antiserum titres of 2048–4096 and 16–128 (reciprocals of dilution end-points) were found for homologous and heterologous antigens, respectively. Cross-reactivity, however, was not obtained in agar immunodiffusion tests when the antigens, in purified preparations or crude infective sap, were treated with pyrrolidine or when the reactants were placed in agar gels containing sodium dodecyl sulphate. Only homologous antigens produced a precipitin line; homologous antiserum titre was 16. In comparative immunodiffusion trials, single-radial-diffusion tests (antiserum incorporated in agar) were much more sensitive than double-diffusion tests for detecting low virus concentrations. Analyses of virus proteins by polyacrylamide-gel electrophoresis demonstrated that coat protein of each virus was composed of a single polypeptide chain with a molecular weight of 35000.     

Serological and immunoelectrophoretic relationships among viruses in the tombusvirus group

Serological cross-reactions among eighteen virus isolates of the tombusvirus group were compared in precipitin tube and immunodiffusion serological tests. The isolates were also compared by immunoelectrophoresis in agar gel. Although precipitin tube tests showed considerable and reproducible differences between the various isolates, the results were too greatly affected by other factors to be of value in assessing strain relationships. When pairs of isolates were compared for spur formation in gel-diffusion tests, the results suggested that most isolates could be placed in one of two groups; one group comprised isolates from pelargonium (leaf curl), the other consisted of petunia asteroid mosaic virus and artichoke mottled crinkle virus isolates from Italy and tomato bushy stunt isolates from soil around this Institute and from cherry. Four isolates did not fall into either of these groups; they nearly always formed spurs when compared among themselves, or with viruses in either of the two groups. Pairs of isolates that could be distinguished from each other in spur-formation tests using antiserum homologous to one of them could not always be differentiated when antiserum heterologous to both isolates was used. Immunoelectrophoresis gave consistent results with several methods of virus preparation; it indicated grouping and separation of the isolates in general agreement with the results of gel-diffusion tests: all pelargonium leaf curl isolates were grouped together with slow migration towards the cathode. The petunia asteroid mosaic isolate and the isolates from cherry and from soil from this Institute (GCRI) moved slowly towards the anode. Tomato bushy stunt virus type strain migrated rapidly to the cathode, differing greatly from all other isolates. The method offers a relatively simple means of typing isolates of the tombusvirus group.     

Complement-Fixation Analysis of Four Subtypes of Foot-and-Mouth Disease Virus Type A

Complement-fixation patterns were established for four subtypes of foot-and-mouth disease virus by block assays against homologous and heterologous antiserum. Inhibition of fixation by excess antigen was observed in most homologous systems but rarely in the heterologous systems. The heterologous antibody titers were, in all instances, considerably lower than those for the homologous systems. Although relatively high dilutions of antiserum may be desirable for subtyping, higher concentrations of antibody should be used for determining serological types.     

Serological relationships and purification of bud necrosis virus,a tospovirus occurring in peanut (Arachis hypogaea L.) in India*

A procedure for the purification of a tospovirus which causes bud necrosis disease (BND) of peanut in India is described. The virus contained three polypeptides of 78 kDa, 54 kDa and 31 kDa. In two ELISA procedures the virus failed to react with antisera to tomato spotted wilt virus (TSWV) obtained from different sources and with an antiserum to impatiens necrotic spot virus (INSV). Additionally, in reciprocal tests TSWV and INSV antigens failed to react with antiserum to the virus infecting peanut in India. In electro-blot immunoassay 54 kDa and 31 kDa polypeptides of the virus reacted with the homologous antiserum. None of the heterologous antisera reacted with any of the three viral polypeptides. On the basis of serological differences the virus that causes BND in India is distinct and therefore has been named bud necrosis virus (BNV). This serotype appears to be restricted to Asia.     

Isolation and Partial Characterization of Peanut Top Paralysis Virus (PTPV), a Destructive Virus Causing a New Disease in Peanut (Arachis hypogaea L.)

A destructive virus, causing top paralysis to peanut, was discovered in the wild germplasm collection growing in the USDA-ARS greenhouses, Stillwater, Oklahoma, USA. The symptoms observed on the wild plant were restricted to a few leaves as green batches in a light green to yellow background with some leaflets having lost most of the basal part of the laminae leaving the top portion rolling upwards forming a cone. The virus was mechanically transmitted to cultivated peanut ( Arachis hypogaea L,.) where it caused more severe and destructive symptoms including stunting, severe malformation of leaves and partial or complete disappearance of leaflet laminae. This virus differed in symptomology, host range, and/or serological reactivity from allpeanut viruses reported in the literature, particularly those causing leaf malformation and stunting. The virus induced necrotic local lesions on Phaseolus vulgaris L. cv. "Topcrop" and chlorotic local lesions with necrotic centres bordered withvery bright intense red color on Chenopodium amaranticolor. In both passive indirect enzyme-linked immunosorbent assay (PAS-ELISA) and Ouchterlony double immunodiffusion test, the virus did not react with antisera against brome mosaic, bean yellow mosaic, peanut stripe, potato Y, tobacco mosaic, watermelon mosaic 1, watermelon mosaic 2, wheat soilborne mosaic, wheat streak mosaic, and zucchini yellow mosaic viruses.
However, in reciprocal cross reactions the virus seemed to share a common antigenic determinant with a peanut mottle virus isolate from Oklahoma (PMV-OK). The virus had flexuous filamentous particles with a length of 750–850 nm, falling within the range reported for the potyvirus group. The virus was successfully purified and the molecular weight of its protein subunit was found to be 30000 d. A polyclonal antiserum was raised in rabbits against the virus and used for reciprocal serological tests.     

Bell Pepper Mottle Virus, a Distinct Tobamovirus Infecting Pepper

Bell pepper mottle virus (BPeMV) can be distinguished by symptomatology and host range from other tobamoviruses but a reliable identification needs serological tests. The relationships of BPeMV to tobacco mosaic virus (TMV), Odontoglossum ringspot virus (ORSV), tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (PMMV) were investigated using precipitin drop tests on slides, immunodiffusion gel tests, double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and indirectELISA using enzyme-linked goat anti-rabbit globulins for the determination of antiserum titers and serological differentiation indices (SDI). Comparisons of SDIs and amino acid composition data demonstrated that BPeMV is a new species of the tobamovirusgroup. BPeMV, ORSV, PMMV, and TMGMV form a cluster within the genus (group) and could be considered as a subgenus of tobamoviruses.     

不同动物制备的抗血清对病毒抗原免疫反应的差异

血清学技术是病毒诊断、鉴定、分类及亲缘关系分析的重要手段。一般常用以制备抗病毒血清的动物是家兔,但也有采用其它动物的,如蛙、羊、豚鼠、鸡及小鼠等。本文比较了Balb/c小鼠、昆明种小鼠和新西兰大白兔对长叶车前花叶病毒上海分离株(RMVsh)和烟草花叶病毒普通株(TMVc)的免疫反应特征。     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

柑橘衰退病毒多克隆和单克隆抗体的制备及检测效果分析

通过改进提纯方法获得了柑橘衰退病毒(Citrustristezavirus,CTV)的提纯液,其产量为1mg/100g植物组织。用CTV免疫大耳白兔,获得多克隆抗体,间接ELISA效价为1∶25600。用CTV免疫小鼠,经细胞融合、ELISA筛选和克隆化培养,获得18株能稳定分泌抗CTV单克隆抗体的杂交瘤单细胞株。对其中4株单克隆腹水抗体进行分析的结果表明,这些抗体的ELISA效价为1∶51200~1∶204800,其中2G和3H的抗体类型及亚类为IgG2a,1E和4H为IgG2b。用所制备抗体对不同来源柑橘样品的CTV检测结果显示,单克隆和多克隆抗体结合使用,采用三抗体夹心ELISA(TAS-ELISA)可以获得理想的检测效果,其特异性强、灵敏度高。同时发现所分析4株单克隆抗体对不同的CTV分离物鉴别能力存在差异,但有关这些CTV分离物的特性及其血清学关系还需进一步研究。     

葡萄卷叶病毒的纯化,血清学研究及其在脱毒组培苗检测中的应用

将具有典型葡萄卷叶病(Grapevine leafroll diseas,GLRD)症状的葡萄组织,经差速和硫酸铯—蔗糖密度梯度离心,提纯了GLRV,并制备了兔抗血清。电镜下可观察到长度从600～2000nm的线形病毒颗粒,其中以1400nm左右为主。免疫电镜结果表明线形病毒颗粒能被美国的NY-1分离株抗血清(Ⅲ型)所修饰。在间接ELISA中提纯制品与GLRV的Ⅲ、Ⅳ、Ⅱ型抗血清均能产生免疫反应。与Ⅲ型抗血清产生较强的免疫反应,Ⅳ型次之,Ⅱ型最弱。在SDS-免疫双扩散实验中病组织韧皮部粗提液与GLRV的Ⅲ,Ⅳ、Ⅱ型抗血清均产生免疫沉淀线。从而推测我国葡萄园内的葡萄卷叶病很可能由2种或3种卷叶病毒感染所致.采用A蛋白夹心酶联免疫吸附试验(PAS-ELISA)检测葡萄试管苗,Ⅲ型抗血清和自制抗血清的平行测试结果基本相符,共获得11个生食葡萄和10个山葡萄品种的脱葡萄卷叶病毒和扇叶病毒的组培苗,扩繁后田间试种表现出良好的农艺性状。     

Relationship between unicellular cyanobacteria established by indirect immunofluorescence of intact cells

Antisera prepared against intact, viable cells were used to show the applicability of a serological approach to detect relationships between unicellular cyanobacteria. Antisera were raised against eight unicellular cyanobacteria and two chlorophycean unicellular organisms. The staining reactivity of each antiserum was tested by the fixed indirect immunofluorescence assay against the different organisms, and each organism was tested for its reactivity with all of the different antisera. Absorption of antisera with the appropriate heterologous antigens was used to further characterize the relationship betweenAnacystis nidulans andSynechococcus cedrorum, and also the relationship between two subcultures of an isolate distinguished by morphological features. Absorption of antiserum was also used for the removal of antibodies to contaminating bacteria. The approaches used are suggested as a useful tool for determining relationships between unicellular cyanobacteria.     

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

Serological cross-reaction between Yersinia enterocolitica O9 and non-O1 Vibrio cholerae bio-serogroup Hakata and antigenic analysis of their relationship by their lipopolysaccharides.

Cross-agglutination and cross-agglutinin absorption experiments were carried out on non-O1 Vibrio cholerae bio-serogroup Hakata (Hakata) and Yersinia enterocolitica O9 (O9). It was shown that the O-antigen of Hakata was closely related to that of O9 in an a, b-a, c type of relationship. The antigenic relationship between the O-antigens of the two bacteria was analyzed by passive hemolysis (PH) and passive hemolysis inhibition (PHI) tests by using their lipopolysaccharides (LPS) as antigen for sensitizing sheep red blood cells (SRBC) and, in the case of the latter, as an inhibitor in a PH system consisting of LPS-coated SRBC, guinea-pig complement and anti-Hakata or O9 antiserum, both unabsorbed and absorbed with the heterologous Hakata or O9 antigen. In the PH experiment, unabsorbed anti-Hakata antiserum had hemolytic titers of 126,100 and 2,600 against Hakata- and O9-LPS-coated SRBC, respectively, and anti-O9 antiserum had hemolytic titers of 19,400 and 38,800, respectively, against these SRBC. The PH experiment showed that anti-O9 antiserum contains a hemolysin reacting with the heterologous Hakata antigen at a high titer (19,400), while anti-Hakata antiserum contains a hemolysin reacting with the heterologous O9 antigen at a significant titer (2,600). The former was completely removed from anti-O9 antiserum with the Hakata antigen and the latter from anti-Hakata antiserum with the O9 antigen. Thus, serological cross-reactivity was demonstrated between the Hakata and O9 strains.     

Passive Hemagglutination-Inhibition Test for Typing Foot-and-Mouth Disease Virus

In addition to currently used serological tests for the occurrence of foot-and-mouth disease virus (FMDV), a specific "passive" hemagglutination-inhibition (HAI) test has been developed as a supplement. Serial twofold dilutions of antiserum (0.05 ml) were mixed with 0.05 ml of a constant concentration of FMDV. After incubating for 30 min at 37 C, agglutinating antibodies were determined by adding 0.1 ml of 2.5% virus-sensitized erythrocytes. The minimum concentration of antiserum required to agglutinate the erythrocytes defined the inhibition in the HAI test. Similar tests using different concentrations of virus to inhibit antibodies were carried out in parallel fashion. The relationship between the logarithm of the HAI titer and the concentration of inhibiting virus was nearly first order (P > 0.25). The slope was used as a measure of the relative specificities of the antigen-antibody interaction and was independent of concentration. The HAI test was type-, subtype-, strain-, and variant-specific with the viral antigens used. In particular, typing was performed directly on bovine antisera.     

Discrimination by gel-diffusion serology of digitaria striate, maize sterile stunt and other rhabdoviruses of Poaceae

Eight rhabdoviruses from grass and cereal hosts and their antisera were used to examine virus relationships by gel-diffusion serology. Nucleocapsid (Nc) preparations from digitaria striate virus (DSV) and maize sterile stunt virus (MSSV) both contained a major protein of c. 52 OOO daltons, and antisera prepared to these readily discriminated related planthopper-transmitted rhabdoviruses. MSSV showed a moderately close relationship to barley yellow striate mosaic virus (BYSMV) when an antiserum prepared to whole virus was used, but the Nc antiserum showed clearer discrimination. Worthern cereal mosaic virus and DSV showed a distant relationship to BYSMV and MSSV. There was no serological relationship between any of these viruses and cereal chlorotic mottle virus, cynodon chlorotic streak virus, festuca leaf streak virus or maize mosaic virus.     

Use of Antibody to Membrane Adenosine Triphosphatase in the Study of Bacterial Relationships

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.     

Natural Infection of Cucumbers by Zucchini Yellow Mosaic Virus in Lebanon

An elongated virus was isolated in the Sin-El-Fil Area east of Beirut from cucumber plants showing severe mottling. The particles were 799 nm long after fixation in glutaraldehyde, but were degraded to shorter pieces in unfixed preparations. Infected cells contained cylindrical inclusions with pinwheel and scroll elements accompanied by proliferated endoplasmic reticulum. The virus was purified and an antiserum was prepared. Different serological tests (slide precipitin test, immunoelectronmicroscopical decoration test, immunosorbent electron microscopy) indicated that it was closely related or identical with zucchini yellow mosaic virus (ZYMV) and more distantly related to watermelon mosaic virus 2 and bean yellow mosaic virus. ZYMV isolates from Italy, France and the Lebanon differ in some host reactions.     

The serological relationships and some other properties of isolates of broad bean wilt virus from faba bean and pea in China

An isolate (N15) of broad bean wilt virus (BB W V) from faba bean in China was compared with some other isolates and strains including the nasturtium ringspot strain (NRSV, BBWV serotype I), parsley virus 3 (PV3, serotype I) and BBWV isolate PV131 (serotype II). In host range studies, N15 infected 12 of 14 species, including soybean and spinach. It was purified from Chenopodium quinoa and pea by a method that yielded up to 8mg/100g tissue. By the same method, NRSV yielded up to 4mg/100 g. Purified preparations of N15 and NRSV contained isometric particles c. 26 nm in diameter which sedimented as three components, N15 at 62, 93 and 117 S, and NRSV at 60, 91 and 116 S. In immunodiffusion tests using antisera to N15 and NRSV, N15 was distinguishable from NRSV but indistinguishable from PV131. In ISEM tests, many more particles of N15 and NRSV were trapped by homologous than by heterologous antiserum; in decoration tests, much antibody attached to homologous particles but none to heterologous particles. In DAS ELISA using N15 antiserum, N15 and six other Chinese faba bean or pea isolates, and a Chinese spinach isolate, were readily detected and were indistinguishable from each other and from PV131; unlike NRSV and PV3, none of the Chinese isolates, nor PV131, was detected using NRSV antiserum. These results indicate that the Chinese isolates belong to BBWV serotype II group.     

水稻条纹病毒病害特异蛋白的提纯及血清学特性

应用差别ｐ Ｈ 值沉淀蛋白质的原理,建立了水稻条纹病毒病特异蛋白（ Ｓ Ｐ） 的两种提纯方法。这两种方法都可以从病叶中提纯到大量的 Ｓ Ｐ,其粗提纯量分别为０ ．８ 和２ ．０ ｍｇ／ｇ 病叶。通过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 分离后得到了精提纯的蛋白,其分子量为２０ ．１ ｋ Ｄａ 。将粗提纯和精提纯的 Ｓ Ｐ 分别免疫兔子,制备出效价为５１ ２００ 和６ ４００ 的抗血清。将效价为６ ４００ 的高度特异性的抗血清用于研究 Ｒ Ｓ Ｖ Ｓ Ｐ 与 Ｒ Ｓ Ｖ Ｃ Ｐ 及同属的水稻草状矮化病毒（ Ｒ Ｇ Ｓ Ｖ） Ｓ Ｐ、 Ｃ Ｐ 之间的血清学关系,结果表明, Ｒ Ｓ Ｖ Ｓ Ｐ 的抗血清与 Ｒ Ｇ Ｓ Ｖ Ｃ Ｐ、 Ｒ Ｓ Ｖ Ｃ Ｐ 之间无反应,但可与 Ｒ Ｇ Ｓ Ｖ Ｓ Ｐ 微弱反应;而 Ｒ Ｇ Ｓ Ｖ Ｓ Ｐ、 Ｃ Ｐ 及 Ｒ Ｓ Ｖ Ｃ Ｐ 的抗血清与 Ｒ Ｓ Ｖ Ｓ Ｐ 之间都无血清学反应。结果证实了 Ｒ Ｓ Ｖ 和 Ｒ Ｇ Ｓ Ｖ 之间存在着进化上的亲缘关系