High temperature inactivation of cucumber and alfalfa mosaic viruses in Nicotiana rustica cultures

Cucumber mosaic (CMV) and alfalfa mosaic (AlfMV) viruses could not be detected in Nicotiana rustica tissues cultured at 32 °C for 16–18 days or at 40 °C for 5 days, but infectivity remained high in comparable tissue cultured at 22 °C. Incubation of infected cultures at 28–30 °C resulted in an initial reduction followed by a partial recovery in the infectivity of both viruses. The infectivity of CMV in tissues grown between 12 and 32 °C was highest in cultures grown at 12 °C. Although CMV infectivity was not detected in cultures after 16–18 days at 32 °C, virus was eliminated only after a further 30 days at 32 °C. When cultures were transferred from 32 to 22 °C after shorter treatment periods, infectivity rapidly increased to levels higher than those of infected tissues grown continuously at 22 °C. At 40 °C, CMV was eliminated from infected tissues after 9 days and AlfMV after 7 days. Cultures grown continuously at 40 °C deteriorated rapidly but, when grown under diurnal alternating periods of 8 h at 40 °C and 16 h at 22 °C, they remained viable and CMV was also inactivated.     

Study of multiplication of cucumber mosaic virus in susceptible and resistant Capsicum annuuum lines

A previous survey on pepper lines (Capsicum annuum L.) indicated that a susceptible cultivar, Yolo Wonder, reacted to cucumber mosaic virus (CMV) by producing a systemic yellow mosaic. By contrast, CMV caused no symptoms on lines Perennial and Vania. The virus is recoverable from the uninoculated leaves of Perennial, while in Vania CMV is restricted to the inoculated leaves. To interpret these phenomena, a comparative study on CMV multiplication rates, yield, specific infectivity and relative proportion of RNAs was made in the inoculated leaves of the three pepper varieties. The rate of CMV multiplication, as estimated by the double antibody sandwich form of enzyme-linked immu-nosorbent assay, was lower in Perennial than in Vania or Yolo Wonder. The yield of virus purified from Perennial was very low when compared with Vania or Yolo Wonder. The specific infectivity of the virus extracted from Perennial was less than that from Vania or Yolo Wonder. These results suggest that Perennial is resistant to CMV multiplication, while restriction of the virus in inoculated leaves of Vania is not due to the inhibition of the virus replication. However, polyacrylamide gel electrophoresis revealed that the RNA profiles of CMV purified from the three pepper lines were similar.     

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.     

Cucumber mosaic virus: viral genes as virulence determinants

TAXONOMIC RELATIONSHIPS: Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus in the family Bromoviridae, which also encompasses the Peanut stunt virus (PSV) and the Tomato aspermy virus (TAV). Nucleotide sequence similarity among these three cucumoviruses is 60%-65%. CMV strains are divided into three subgroups, IA, IB and II, based on the sequence of the 5' untranslated region of the genomic RNA 3. Overall nucleotide sequence similarity among CMV strains is approximately 70%-98%. GEOGRAPHICAL DISTRIBUTION, HOST RANGE AND SYMPTOMATOLOGY: CMV is distributed worldwide, primarily in temperate to tropical climate zones. CMV infects more than 1200 species of 100 plant families, including monocot and dicot plants. Symptoms caused by CMV infection vary with the host species and/or CMV strain, and include mosaic, stunt, chlorosis, dwarfing, leaf malformation and systemic necrosis. CMV disease is spread primarily by aphid transmission in a nonpersistent manner. PHYSICAL PROPERTIES: In tobacco sap, the thermal inactivation point of the viral infectivity is approximately 70 °C (10 min), the dilution end-point is approximately 10(-4) and viral infectivity is lost after a few days of exposure to 20 °C. Viral infectivity can be retained in freeze-dried tissues and in the form of virions purified using 5 mm sodium borate, 0.5 mm ethylenediaminetetraacetic acid and 50% glycerol (pH 9.0) at -20 °C. CMV particles are isometric, approximately 28-30 nm in diameter and are composed of 180 capsid subunits arranged in pentamer-hexamer clusters with T= 3 symmetry. The sedimentation coefficient (s(20) ,(w) ) is c. 98 S and the particle weight is (5.8-6.7) × 10(6) Da. The virions contain 18% RNA. The RNA-protein interactions that stabilize the CMV virions are readily disrupted by sodium dodecylsulphate or neutral chloride salts. GENOMIC PROPERTIES: The genomic RNAs are single-stranded messenger sense RNAs with 5' cap and 3' tRNA-like structures containing at least five open reading frames. The viral RNA consists of three genomic RNAs, RNA 1 (c. 3.3 kb), RNA 2 (c. 3.0 kb) and RNA 3 (c. 2.2 kb), and two subgenomic RNAs, RNA 4 (c. 1.0 kb) and RNA 4A (c. 0.7 kb). The 3' untranslated regions are conserved across all viral RNAs. CMV is often accompanied by satellite, noncoding, small, linear RNA that is nonhomologous to the helper CMV.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Rubella Virus: Continuous and Concurrent Production of Complement-fixing Antigen and Infectious Virus in Suspension Cultures

Rubella complement-fixing (CF) antigen and infectious virus were produced continuously and concurrently for as long as 63 days in suspension cultures of BHK-21 cells prepared from uncloned monolayer stock cultures. CF titers ranged from 1:4 to 1:32, and the peak infectivity titer was greater than 8.0 (TCID(50) log(10)) per ml. Suspension cultures could be recultivated after prolonged storage in liquid nitrogen. The resulting monolayer or suspension cultures also produced CF antigen. Suspension cultures provide an effective system for the long-term continuous and concurrent production of rubella virus diagnostic reagents.     

Indices of water deficit in susceptible and resistant cucumbers in response to infection by cucumber mosaic virus

Indices of water deficit were determined under conditions of non-limited water supply in cucumber mosaic virus (CMV)-infected seedlings of the susceptible cv. Bet Alpha. An increase in the concentration of soluble solids, decrease of water and osmotic potentials, and increase of proline concentration were found in the CMV-infected cotyledons. In the cv. Shimshon, which is resistant to CMV, virus infection caused only a slight change in the concentration of the soluble solids and in the osmotic potential; water potential and proline content were not affected. Concomitantly, infectivity of cotyledons by CMV was much lower in the tolerant cv. Shimshon than in the susceptible cv. Bet Alpha. The possible association of water deficit with virus-induced growth retardation is discussed.     

带卫星RNA黄瓜花叶病毒保护接种的植物中病毒积累与基因组RNA合成及病状表达

本文报道了三生烟在接种带卫星RNA的黄瓜花叶病毒(CMV-S52)后,或接种后用强毒CMV攻击,其病状表现、组织中病毒含量,病毒基因组RNA合成与卫星RNA合成的关系.植物在接种CMV-S52后7～10天,所有接种植物都表现出不同程度的轻度花叶症状,此期正是组织内病毒含量最高,而卫星dsRNA占总dsRNA百分比较低时期.接种后不同时间的测定证明,组织内病毒基因组dsRNAl和dsRNA2的合成水平均较低,而卫星dsRNA则始终保持较高的合成水平;到接种后15天病状消失时,卫星dsRNA占总dsRNA合成百分比的84.79％之多. 用CMV-S52保护接种后再用强病毒攻击,植物中的病毒含量、病毒ssRNA合成不增加或稍有增加;而基因组dsRNA和卫星dsRNA合成都有增加,但卫星dsRNA合成仍占绝对优势.保护作用的效果,取决于攻强病毒时已存在于组织内的卫星RNA与基因组RNA合成量的相对比例。讨论了卫星RNA的保护机理。     

Studies on haemagglutination by the EMC-MM-Columbia SK-group of viruses

Summary When MM and Columbia SK virus were propagated in the chick embryo, the virus disappeared from the allantoic fluid and from the embryonic brain tissue after 6 to 9 chorioallantoic passages. Amniotic passages, however, resulted in a gradual increase of the mouse infectivity titre of the amniotic fluid and of the fluid of the regularly observed subcutaneous edema of the embryos, though after 22 passages the ID50 titre was still 3 logs lower than that of the original mouse brain suspension. The virus was present not only in amniotic fluid, but also in allantoic fluid and throughout the whole embryo. In spite of the pretty high mouse infectivity titres, the extra-embryonic fluids failed to produce haemagglutination of sheep red cells, whereas low haemagglutination titres could be obtained with suspensions of embryonic brain tissue. No evidence was obtained, that a non-specific inhibitor was responsible for the inability to produce haemagglutination. The virus present in egg fluids is only partially adsorbed, which is in contrast with the almost complete adsorption of virus in mouse brain suspension. The adsorption of a relatively small amount of virus might be one of the factors responsible for lack of haemagglutinating ability. It is suggested, that another coinciding factor, which is necessary for the initiation of the haemagglutination might be present in the central nervous system lesion.Third publication: Antonie van Leeuwenhoek17, 137, 1951.     

Antigenic cross-reactions among herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus.

Polyvalent rabbit antisera against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV), monospecific antisera against affinity-purified HSV-2 glycoproteins gB and gG, and a panel of monoclonal antibodies against HSV and EBV proteins were used to analyze cross-reactive molecules in cells infected with the four herpesviruses. A combination of immunoprecipitation and Western blotting with these reagents was used to determine that all four viruses coded for a glycoprotein that cross-reacted with HSV-1 gB. CMV coded for proteins that cross-reacted with HSV-2 gC, gD, and gE. Both CMV and EBV coded for proteins that cross-reacted with HSV-2 gG. Antigenic counterparts to the p45 nucleocapsid protein of HSV-2 were present in HSV-1 and CMV, and counterparts of the major DNA-binding protein and the ribonucleotide reductase of HSV-1 were present in all the viruses. The EBV virion glycoprotein gp85 was immunoprecipitated by antisera to HSV-1, HSV-2, and CMV. Antisera to CMV and EBV neutralized the infectivity of both HSV-1 and HSV-2 at high concentrations. This suggests that cross-reactivity between these four human herpesviruses may have pathogenic as well as evolutionary significance.     

Adaptation of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> soybean strains (SSVs) to cultivated and wild soybeans

Cucumber mosaic virus soybean strains formerly called soybean stunt virus (SSV) were inoculated onto 23 wild soybeans collected from four Asian countries to investigate their infectivity in order to improve understanding of the co-evolution of SSVs and soybean. SSV inoculation resulted in systemic infection in most of the wild soybeans used. However, an SSV strain (SSV-In), which was isolated in Indonesia, did not result in systemic infection of many of the wild soybeans distributed in southern Japan. This exceptional infectivity of SSV-In may be due to its specific adaptation to the local soybean population(s) of Indonesia, which has rarely been affected by gene flows from wild soybean. In the present study, the nucleotide sequences of the 3a and CP genes of SSV were determined, and the data were used to classify seven SSV isolates among known Cucumber mosaic virus (CMV) strains. The phylogenetic analysis showed that the seven SSVs formed a distinct cluster separated from the other CMV strains despite their different geographical origins; SSV-In was the most divergent of the seven isolates. Comparison of the rates of synonymous and nonsynonymous substitutions revealed that the SSV group had evolved faster than subgroup IA. The implications of the findings are discussed in relation to the so-called Red Queen hypothesis.     

Effect of proteins on reovirus adsorption to clay minerals.

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

An Isolate of Cucumber Mosaic Virus from Fluted Pumpkin in Nigeria

The properties of a virus isolated from fluted pumpkin (Telfairia occidentalis) was investigated. It produced symptoms in some members of the Solanaceae and Leguminosae, and was transmitted nonpersistently by the aphid, Aphis spiraecola. On the basis of these alone, it is distinct from another previously described virus, Telfairia mosaic virus, which neither caused symptoms in members of these families nor was it transmitted by insects. Furthermore, the virus in crude sap or purified preparations reacted with antiserum to cucumber mosaic virus (CMV), but not with antisera to several common viruses in Nigeria. Electron microscopic examination revealed isometric particles of 29 × 1 nm in diameter. These properties confirmed that the virus is an isolate of CMV, and closely resembles the Y-strain in causing systemic mosaic symptoms in Vigna unguiculata. From infectivity and pathogenicity tests, it is concluded that it is the main cause of mosaic disease in fluted pumpkin.     

Effect of proteins on reovirus adsorption to clay minerals

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Plant resistance to fungal diseases induced by the infection of cucumber mosaic virus attenuated by satellite RNA

Biological control agents (BCAs) composed of attenuated cucumber mosaic (CMV) and its satellite RNA for controlling CMV diseases were found to induce plant resistance to a number of fungal diseases. Tests conducted in both the field and greenhouse showed evident protective effects against fungal infections by the BCAs. Artificial inoculation with a fungal spore suspension using BCA-treated plants, satellite transgenic plants and plants infected with CMV alone indicated that the resistance to fungi was induced by the virus infection, not by the presence of satellite RNA.     

Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.     

Cytomegalovirus-induced membrane antigens in productively infected cells.

Adsorbed but not penetrated virus can be removed from the CMV-infected cell membrane by digestion with cystine-activated papain. Membrane antigens appear on 80-90% of the infected cells 14-20 hr after infection as a result of de novo protein synthesis. Antigen synthesis can be blocked with inhibitors of protein synthesis, but not with DNA inhibitors. In the early stage of infection, pooled human convalescent serum reacted well with the membrane antigen, whereas pooled antiserum of rabbits immunized with CMV virion suspension gave a positive reaction with a small proportion of the cells. After the 48th hr, both the human and the rabbit serum pool reacted with the membrane of the infected cells. Absorption with cell cultured for 24 hr after CMV infection reduced the neutralization titres of the antisera only slightly but the titre reduction was considerable when absorption was performed with cells cultured for more than 48 hr after infection. It is concluded that on the membrane of cells productively infected by CMV at least two membrane antigens are present, one coded for by the DNA of the parent virus and another which is the product of the DNA of the virus progeny. The two antigens can be differentiated serologically.     

A Cucumber Mosaic Virus Based Expression System for the Production of Porcine Circovirus Specific Vaccines

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.