Grapevine Fanleaf Virus Monoclonal Antibodies: their Use to Distinguish Different Isolates

Monoclonal antibodies (MCAs) specific for Grapevine Fanleaf Virus (GFV) were obtained by fusion of a non-secreting myeloma cell line with the spleen cells from Balb/c mice immunized with GFV isolate F 13. The MCAs were tested with apanel of GFV isolates, using both crude extracts and purified virus particles. Some of the MCAs recognize all 41 isolates tested and are suitable as a diagnostic reagent for GFV detection. Certain MCAs do not bind to some of the isolates, while others show a lower reactivity for certain isolates than for the immunogen F 13. MCAs make it possible to distinguish GFV isolates which until now could not be distinguished by using polyclonal antiserum.     

An Electron Microscope Study of Tubules in Homogenates of Chenopodium quinoa Infected with Strawberry Latent Ringspot Virus

Tubular structures found in tissue homogenates of Chenopodium quinoa, infected with a parsnip isolate of SLRV, were described. Virus-like particles within the tubules had been differentially penetrated by “negative” stain. The presence within some tubules of particles fully penetrated by stain was apparently a new observation for SLRV.     

生物素标记GFV－cDNA探针的制备及在检测葡萄扇叶病毒上的应用

以整合到质粒中的ＧＦＶ－ｃＤＮＡ为模板经ＰＣＲ合成了生物素标记的ＧＦＶ单、双链探针。用合成的探针对提纯的ｃＦＶ－ＲＮＡ＿２、感染ＣＦＶ的昆诺藜叶及１８株而萄进行ＤＮＡ－ＲＮＡ杂交检测表明：检测提纯病毒ＲＮＡ＿２的灵敏度为１．５ｐｇ／斑点,感染ＧＦＶ的昆诺藜提取液最高稀释度可达４０９６０倍;１１株显示典型扇叶症状的样品杂交结果均为阳性,且汁液稀释４００～８００倍仍能测出,７株不显示典型扇叶症状的葡萄中３株受到ＧＦＶ的侵染。单、双链探针最适使用浓度分别为１／２００及１／１００。     

An Electron Microscope Study of Polyoma Virus in Hamster Kidney

Electron microscope studies were made of hamster kidneys taken at daily intervals after injection of a variant of polyoma virus into newborn animals. Particular attention was paid to the period 5 to 6 days after injection at which time the necrotizing response was at its peak and virus particles were seen in greatest numbers. The most numerous particles were about 28 mµ in diameter. They were observed mainly within nuclei of stromal cells and are similar to the particles seen in large numbers in polyoma-infected mouse cells growing in vitro. They were not observed in cells of fully developed tumors. Filamentous or tubular structures closely associated with the 28 mµ particles and probably concerned in their formation are described. Considerable quantities of viral material were contained within cytoplasmic inclusions. In some of the inclusions larger particles of diameter 60 mµ were observed. The origin of these particles and their relation to the 28 mµ particles is discussed.     

Effects of Grapevine Fanleaf and Stem Pitting Viruses on the Photosynthetic Activity of Grapevine Plants Grown in vitro

The effects of viral diseases on the photosynthetic activity of grapevine (Vitis rupestrisvar. Rupestris du Lot) leaves were investigated. The third and sixth leaves used for measurements were obtained from in vitrogrown healthy plants and plants affected by grapevine fanleaf and rupestris stem pitting viruses. The induction curves of prompt and delayed chlorophyll fluorescence, as well as the temperature characteristics of steady-state, prompt, and delayed emissions, were investigated. Age-dependent changes were found, which were related, on the one hand, to the acceleration of electron transport and the enhancement of thylakoid energization and, on the other hand, to a smaller extent of transmembrane H⁺in the younger sixth leaf compared to that in the third leaf. The infected plants characteristically showed faster electron transport, an elevated energetic efficiency of photosynthesis, and the suppression of CO₂fixation owing to a presumable activation of the adenylate metabolism. An analysis of the thermograms of prompt and delayed fluorescence revealed the shifts in the position of the M ₁peak and a half-inhibition temperature T₅₀towards a higher temperature in infected plants, which indicates a certain increase in the thermal tolerance of thylakoid membranes. The data suggest that the viral metabolism affects the functional activity and stability of thylakoid membranes.     

Molecular Characterization of Whole Genomic RNA2 From Iranian Isolates of Grapevine Fanleaf Virus

Grapevine fanleaf virus (GFLV) is the major causal agent of the grapevine degeneration disease. To characterize the genomic RNA2 segment from Iranian isolates of GFLV, leaf samples were collected from infected vineyards in different locations with a long history of vine cultivation. Four isolates were selected for cloning and sequencing on the basis of the restriction profiles of RT‐PCR products. The sequencing data revealed that the RNA2 of the Iranian GFLV isolates were the shortest compared with that of all previously described GFLV isolates. The sizes were 3730 nucleotides (nt) for Shir‐Amin and Urmia isolates and 3749 nt for Takestan and Bonab isolates (excluding the poly (A) tail), due to deletion events in both 5′ and 3′ non‐coding regions. In the phylogenetic tree based on the full‐length nucleotide sequences of GFLV RNA2, all the GFLV isolates clustered into two groups with the exception of the Hungarian isolate (GHu). The Iranian isolates grouped as a distinct cluster. Recombination analyses showed that GFLV‐NW (Germany), GFLV‐F13 (reference isolate), GFLV isolate Shir‐Amin (Iran) and Arabis mosaic virus isolate Lv were recombinant isolates and one of their parents belonged to the same lineage as the Iranian isolates. These findings suggest that these isolates originated from a common ancestor.     

不同CMV分离物侵染寄主的超微结构变化

应用电镜观察了黄瓜花叶病毒CMV不同分离物侵染寄主的细胞 超微结构变化。来自一串红(Salvia splendens)的不含卫星RNA分离物M-22侵染心叶烟,病毒粒子散布于细胞质,在液泡中形成大片病毒粒子结晶,液泡膜边缘产生小泡结构,完整的病毒粒子穿过胞间连丝在细胞间运转,胞间连丝中央部分有扩张现象。自然感染三生烟的含坏死卫星RNA分离物8-S1侵染普通烟,病毒粒子分散于细胞质,在液泡中未观察到结晶体,叶绿体产生囊泡结构,部分病毒粒子处在叶绿体空泡中。田间寄主上受8-S1侵染的三生烟细胞质中分布着大量球形病毒粒子,叶绿体也产生含有病毒粒子的囊泡结构。表明含有卫星RNA和不含卫星RNA的CMV分离物引起的细胞病变特征存在差别,可能是CMV卫星RNA参与病理变化的依据之一。     

不同CMV分离物侵染寄主的超微结构变化

应用电镜观察了黄瓜花叶病毒ＣＭＶ不同分离物侵染寄主的细胞超微结构变化。来自一患红（Ｓａｌｖｉａｓｐｌｅｎｄｅｎｓ）的不含卫星ＲＮＡ分离物Ｍ－２２侵染心叶烟,病毒粒子散布于细胞质,在液泡中形成大片病毒粒子结果,液泡膜边缘产生小泡结构,完整的病毒粒子穿过胞间连丝在细胞间运转,胞间连丝中央部分有扩张现象。     

An Electron Microscope Study of Myelin Figures

In the electron microscope, thin sections of OsO₄-fixed myelin figures from the phospholipide fraction of human brain show a pattern of parallel dark lines with a repeating period of about 40 A. It is shown that the dark lines probably represent the reaction product of OsO₄ with double bonds in the fatty acid chains, thereby marking the central portion of one bimolecular lamella. The addition of globin results in dense lines 25 to 50 A wide that cover the surface of the myelin figures. When such a figure consists of only two bimolecular leaflets of lipide covered with globin, the structure shows striking similarity to the image of cell membranes in fixed tissue sections. A hypothetical schema is given of the molecular structure of the figure, and the distribution of OsO₄ in it.     

Biotin-Avidin ELISA Detection of Grapevine Fanleaf Virus in the Vector Nematode Xiphinema index

The value of biotin-avidin (B-A) ELISA for the detection of grapevine fanleaf virus (GFLV) in Xiphinema was estimated with field populations and greenhouse subpopulations. Samples consisted of increasing numbers of adults ranging from 1 to 64 in multiples of two. Tests with virus-free X. index populations reared on grapevine and fig plants as negative controls did not reveal a noticeable effect of the host plant. ELISA absorbances of virus-free X. index samples were greater than corresponding absorbances of X. pachtaicum samples. Differences occurred between two X. index field populations from GFLV-infected grapevines in Champagne and Languedoc. In most tests, 1-, 2-, 4-, and 8-nematode samples of virus-free and virus-infected populations, respectively, could not be separated. Consequently, B-A ELISA was not a reliable method for GFLV detection in samples of less than 10 X. index adults, but comparison of the absorbances obtained with increasing numbers may allow differentiation of the viral infectious potential of several populations.     

An Electron Microscope Study of the Rat Ovum

This paper reports on the fine structure of rat oocytes at stages before ovulation, during maturation, fertilization, and early cleavage. The study includes parallel observations on light and electron microscope preparations with attempted correlations. The follicular cells of the ovarian egg are described as sending long processes through the zona pellucida to the egg surface where they mingle with thin projections from the egg itself. No open communication between follicle cell cytoplasm and egg cytoplasm was observed. During maturation and fertilization both types of processes are withdrawn from the zona. The germinal vesicle and later the pronuclei of the fertilized egg are characterized by numerous large nucleoli. These have the form of thick walled vesicles with diameters as great as 8 to 10 µ. The wall is dense in the EM image and appears to consist in part of small granules. The cytoplasm shows several inclusions including mitochondria of usual form and a Golgi component which has the typical fine structure and the distribution described by earlier light studies. Small dense particles, presumably RNP particles, are distributed throughout the cytoplasmic matrix and show no preference for membranes. The endoplasmic reticulum of the oocyte is represented by a scattering only of vesicles, but begins a more extensive and elaborate development with the onset of segmentation. One inclusion of the ooplasm, similar in size to mitochondria, receives special attention. It is a vesicular structure, containing a large number of small vesicles (10 to 50 mµ in diameter) and frequently a central density or nucleoid. They are referred to as multivesicular bodies. Such bodies are found in small number in the ovarian egg, but increase greatly in number during maturation and fertilization. It appears from the micrographs of eggs in these latter stages that these vesicular bodies break down and liberate their content of small vesicles to the surrounding ooplasm. Comments are provided on the apparent significance of the various observations.     

Electron Microscopic Study of Hemadsorption on Vaccinia Virus Infected Cells

Hemadsorption (HAD) induced in HEp-2 cells infected with vaccinia virus was observed. In ultrathin sections, binding of 36 red blood cells (RBCs) was examined in detail and 3 types of HAD were observed: (1) direct and close binding of RBCs to infected HEp-2 cells (cyto-HAD) was observed in cross sections of 27 RBCs, (2) binding of RBCs through microvilli of infected cells was found in 11 RBCs, and (3) five RBCs were distorted to form tentacle-like projections by which they were bound to the HEp-2 cell surface. Scanning electron microscopy revealed that more than 30% of the RBCs were bound to microvilli of vaccinia virus-infected HEp-2 cells, and that the number of microvilli twined round each RBC was over ten. RBCs were attached to certain microvilli through swollen sucker-like tips which were not observable in non-infected HEp-2 cells. RBCs sometimes revealed a polygonal shape at regions of binding to microvilli. Virion-mediated RBC-HEp-2 cell binding could not be observed.     

An Electron Microscope Study of the Spermatozoid of Sphagnum

The micro-anatomy of the cilia of Sphagnum has been elucidatedin greater detail than in the ciliary types previously investigated.The facts are summarized in a diagrammatic reconstruction.     

An Electron Microscope Study of Cysts in the Saprolegniaceae

The structure of the zoospore cysts of various members of theSaprolegniaceae has been studied by electron microscopy. InSaprolegnia ferax, S. dioica, in Isoachlya eccentrica and I.unispora the primary cysts were smooth, whilst the secondarycysts bore stalked double-headed hooks. In S. parasitica thesecondary cysts bore tufts of longer hooks. In Protoachlya,Achlya, and Brevilegnia neither type of cyst bore hooks. InDictyuchus sterile the secondary cysts bore large spiny projections.The primary cysts in many species bore tufts of radiating hairs,and it is suggested that these are the remains of ciliated flagella.     

Ultrastructural Alteration of Host Plants Infected with Tomato Mosaic Virus

The ultrastructural aheration of two host plants infected with tomato mosaic virus (ToMV) were studies with transmission electron microscopy. A large number of virus particles were found being accumulated in different cells such as epidermis, parenchyma cells and vascular bundle cells of Lycopersicon esculentum Mill. grown at 25℃ Crystalline inclusions and paracrystal inclusions composed of ToMV particles were observed in the cytoplasm or vacuoles. Some muhivesicular bodies and myeloid bodies protming into the vacuole and vires-specific vesicles associated with the tonoplast were also observed. The ultrastructuml alteration of Nicotiana tabacum L. tv. Xanthinn was similar to that in tomato infected by ToMV grown at 25 cE. In addition to the aggregate inclusions described above, some cytoplasmic angularly-layered aggregates and abnormal chloroplasts with small peripheral vesicles were observed in the parenchyma cells. The densely stained amorphous material was seen in the cytoplasm of N. tabacum L. cv. Xanthiun grown at 35℃. No X- body was observed in the cytoplasm of the ToMV infected tomato and tobacco grown at 25℃ or 35℃. The authors' results suggest a significant difference between the cytopathological effects of ToMV and tobacco mosaic virus (TMV). These characteristic difference may be useful in the virus diagnosis and identification virus infections in plants.     

Replication of Sindbis Virus IV. Electron Microscope Study of the Insertion of Viral Glycoproteins into the Surface of Infected Chick Cells

The appearance of Sindbis virus-envelope glycoproteins in the surfaces of chicken embryo fibroblasts was studied by an indirect labeling technique. This technique involved treating infected cells sequentially with rabbit immunoglobulin G (IgG) specific for Sindbis virus followed by hemocyanin-conjugated goat (anti-rabbit IgG) IgG; surface replicas of these cells were then prepared and examined in the electron microscope. As early as 2 h after infection (and at least 1 h before mature virions were released), newly synthesized virus-envelope glycoproteins were detected at the cell surface. By 3 h after infection, cell surface membranes were extensively modified by the insertion of the Sindbis glycoproteins. When infected cells were prefixed with glutaraldehyde before labeling, the glycoproteins were distributed fairly evenly over the cell surface, although a slight clustering was observed on cells labeled early in infection. However, no evidence for large-scale clustering of virus glycoproteins corresponding to patches of budding virus was observed. Similar results were found with unfixed cells labeled at 4 C. However, when unfixed cells were labeled at 37 C, the glycoproteins were shown to be in discrete clusters, demonstrating that these glycoprotein antigens can diffuse laterally through the cell membrane at this temperature.