- Volume 46, Issue 1, 1980
Volume 46, Issue 1, 1980
- Bacterial
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The Effect of Temperature on the Ecology of Aquatic Bacteriophages
More LessSUMMARYThree physiological types of coliphage were recognized on the basis of the effect of temperature on their e.o.p. High temperature (HT) phages plated at or above 25 °C, low temperature (LT) phages at or below 30 °C and mid-temperature (MT) phages in the range 15 to 42 °C. Only LT phages were found for Aeromonas hydrophila, an indigenous water organism. The maximum and minimum plating temperatures were stable properties of the virus and were not influenced by the growth temperature of the host. Temperature was found to affect the adsorption of two phages and to affect multiplication, but not adsorption, of another two. The distribution of the three types of phage correlated closely with the temperature of the environment from which they were isolated. The ecological implication of these results is discussed.
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- Animal
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The In Vivo Differentiation of Strains of Yellow Fever Virus in Mice
More LessSUMMARYStrains of yellow fever virus isolated since 1927 in Africa and the Americas, and strains derived from them, have been differentiated by the responses of mice of different ages to intraperitoneal (i.p.) or intracerebral (i.c.) infection. Infection, antibody conversion, protection and death have been presented on age-dose response phase diagrams that serve as in vivo ‘fingerprints’ for the differentiation of virus strains and their modifications through passage and selection. Correlations between marker characteristics are discussed in terms of the efficiency of infection, regulatory (pre-challenge) and protective (post-challenge) immunity, and the expression of virulence. The requirement in virus strain specification for the resolution of events on pathogenic and immunogenic pathways is discussed.
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A Comparison of the Structural Polypeptides of Five Strains of Mumps Virus
More LessSUMMARYThe polypeptide patterns of five strains of mumps virus have been compared under uniform electrophoresis conditions. These strains included the MJ and RW strains, recently isolated from human cerebrospinal fluid, the neuro-adapted Kilham strain and the chick embryo-adapted Enders and Jeryl Lynn strains. A similar pattern of polypeptides, featuring six common and virus-specific species, was obtained with purified virus grown in ovo, in chick embryo fibroblasts (CEF) or in Vero cells. These polypeptides have been designated VP1 (mol. wt. 200000), VP2 (mol. wt. 80000 or 75000), VP3 (mol. wt. 68000), VP4 (mol. wt. 58000), VP5 (mol. wt. 45000), VP6 (mol. wt. 39000). The virus glycoproteins were identified as VP2 and VP4; the nucleocapsid-associated proteins as VP1, VP3 and VP5. Additional polypeptides occurring both in uninfected Vero cells and in uninfected CEFs appeared in polypeptide profiles of purified mumps virus preparations, and could not be designated virus-specific polypeptides. Although strain differences were minimal, we did observe (1) an extra protein, nucleocapsid-associated, in patterns from the chick-adapted Enders strain of mumps virus and (2) strain-dependent variability in the size of VP2.
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Comparison of Three Methods of ELISA for Baculoviruses
More LessSUMMARYDirect, indirect and double antibody sandwich methods of ELISA were examined for their ability to detect and discriminate between granulosis viruses from Pieris brassicae, Agrotis segetum and Cydia pomonella and for their specificity in the presence of host material. The indirect method was the most sensitive, capable of detecting down to about 1 ng of dissolved capsules/ml compared with 10 ng/ml for the double antibody sandwich method and 25 ng/ml for the direct method. The double antibody sandwich method showed greatest discrimination between different granulosis viruses; the direct method was not quite so specific and the indirect method varied considerably depending upon the antibody concentration used although even with this latter method cross-reactions were restricted to baculo-viruses and did not occur with occluded insect viruses in other taxonomic groups. All three methods were highly specific in the presence of large amounts of host material and could be used to assay virus in diseased larvae, although at very high levels of contamination, equivalent to adding 100 healthy larvae to one diseased larva, only the double antibody sandwich method could still reliably detect virus though with reduced absorbance readings. Possible reasons why the ELISA shows much greater discrimination between different Baculovirus inclusion bodies than other serological techniques are discussed.
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Acquisition of Susceptibility to Coxsackievirus A2 by the Rat L8 Cell Line During Myogenic Differentiation
More LessSUMMARYCoxsackievirus A2 was propagated to high titres in the established rat L8 myogenic cell line during the stage of differentiation characterized by fusion of myoblasts into multinucleated myotubes (192 h post-plating cultures). In contrast, pre-fusion mononucleated L8 cultures infected 24 h after plating were refractory to Coxsackievirus A2 infection, as was a non-fusing clonal variant of the L8 line (L8CL3-U) regardless of the age of the latter cultures. The development of virus susceptibility in the older L8 cultures correlated with a marked virus-specific c.p.e. as evidenced by vacuolated degenerating myotubes with disrupted cytoplasm, whereas no c.p.e. was seen following infection of the young L8 or variant L8CL3-U cultures. The restriction of Coxsackievirus A2 replication in young L8 and L8CL3-U cultures occurred after virus attachment.
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Comparison of Foldback Sequences of Herpes Simplex Virus Types 1 and 2 DNA
More LessSUMMARYThe DNAs of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) were separately denatured and allowed to renature briefly. The intrastrand foldback structures that resulted from base pairing of inverted repeated sequences on otherwise single-stranded (ss) DNA were visualized in the electron microscope. The two genomes were found to contain similar size classes of small duplex stem DNA sequences. However, HSV-2 DNA appeared to possess an additional, larger size class of foldback structures not found on HSV-1 DNA. Both HSV DNAs were found to contain stem-plus-loop structures; the larger stem-plus-loop structures of the two genomes had similar stem lengths but dissimilar loop lengths. Thus, a comparison of the genomes of HSV-1 and HSV-2 showed that they possessed similar size classes of foldback sequences.
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Host Antigens on Avian Oncoviruses: Evidence for Virus Envelope Antigens Related to Specific Chicken Erythrocyte Membrane Antigens
More LessSUMMARYAvian sarcoma viruses (ASV) of subgroups A to D, produced by chick embryo fibroblasts (CEF), are inactivated to a high degree by rabbit antisera to the membrane antigens of adult chicken and chick embryo erythrocytes, notably by antisera to an antigen of embryo erythrocytes, which is lost by adult erythrocytes and to another antigen specific to the latter erythrocytes. Contrary to virus inactivation by anti-CEF serum reported earlier, virus inactivation by the antisera to these two age-specific antigens does not require complement and is not paralleled by virolysis but by aggregation of virions.
The two antigens related, or identical, to the age-specific erythrocyte membrane antigens thus shown to be present on the virus envelope do not pre-exist, or pre-exist only in a low amount, on the CEF membrane, since the virus-inactivating capacity of their antisera is not removed by absorption with CEF. Their appearance on the virus does not depend on cell transformation but only on infection, since both antigens are found on a ts ASV mutant produced at restrictive temperature by untransformed CEF and the virus-inactivating capacity of their antisera is removed by absorption with CEF infected with Rous-associated virus (RAV-1). These findings suggest that infection of CEF by avian oncoviruses may elicit the appearance, or enhance the expression at the cell surface of antigens characteristic of another cell type which may contribute to the formation of specific virus budding sites.
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Ultrastructural Study of Rotavirus Replication in Cultured Cells
More LessSUMMARYA systematic ultrastructural analysis of the replication cycle of the simian rotavirus SA11 in permissive MA104 cells was performed under reproducible conditions. At 8 h p.i., small areas of viroplasm were seen adjacent to swollen vesicles of the rough endoplasmic reticulum (rer) containing a few 80 to 90 nm virus particles. At later times, the size and number of these inclusions increased and the rer contained large numbers of the 80 to 90 nm particles as well as 52 to 65 nm particles. Infected cells eventually lysed, releasing progeny virus. Other cytological alterations included virus particles sequestered in lysosome-like bodies, 15 to 20 nm tubular structures in the nucleus and/or cytoplasm, convoluted membranes within the rer, filament bundles associated with virus particles, and mitochondria containing 1 to 5 virus particles. In addition, SA11 replication was studied in several less permissive cell lines. The results were similar to those with MA104 cells except that a smaller percentage of the cells were productively infected.
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Effect of Interferon on Mouse Leukaemia Virus (MuLV). V. Abnormal Proteins in Virions of Rauscher MuLV Produced in the Presence of Interferon
More LessSUMMARYInterferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of non-infectious particles (‘interferon’ virions) containing the structural proteins coded by the env and gag genes as well as additional virus polypeptides. The major glycoprotein detected in the control virions is gp71, but ‘interferon’ virions contain in addition an 85K mol. wt. (gp85) glucosamine-containing, fucose-deficient glycoprotein. This is recognized by antiserum to MuLV and may be related to env pr85. Surface iodination of intact virions indicates that gp71 and gp85 are the two major components of the external envelope. However, whereas in control virions gp71 associates with p15E (gp90), this complex was not detected in ‘interferon’ virions. Analysis of radio-labelled (3H-amino acids or iodinated) proteins from disrupted ‘interferon’ virions revealed the presence of 65K, 55K, 40K, 20K and 12K mol. wt. polypeptides which could be precipitated with antiserum against MuLV. There was a distinct difference in the patterns of incorporation of pulse-labelled 3H-amino acid polypeptides into virions in the presence and absence of interferon. Those polypeptides labelled in the presence of interferon and recovered in the extracellular virions in a chase with interferon appeared to have substantially fewer copies of p30 and more of gag pr55 polypeptide than the controls. These results indicate that in the presence of interferon there are changes in the proteolytic cleavage associated with virion assembly.
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Attempts to Induce Interferon Production by IdUrd Induction and EBV Superinfection in Human Lymphoma Lines and their Hybrids
More LessSUMMARYInducers of the Epstein-Barr virus (EBV) cycle, 5-iodo-2-deoxyuridine (IdUrd), phorbol myristate acetate (TPA) and sodium butyrate were tested for their ability to induce EBV-determined antigens, early antigen (EA) and virus capsid antigen (VCA), and to stimulate interferon (IF) production in a variety of EBV-carrying lymphoid cell lines. IdUrd and TPA induced IF production to various extents in the different lines, whereas sodium butyrate did not. There was no relationship between induction of the EBV cycle and production of IF; the two appear to be independent characteristics.
Superinfection with the transforming B95-8 virus substrain of EBV induced IF production, whereas superinfection with the abortively cytopathic, non-transforming P3HR-1 substrain had little or no IF-inducing effect, in spite of its highly potent effect on virus antigen (particularly EA) synthesis.
Analysis with specific antisera against IF showed that IF preparations produced by three different lymphoid cell lines in response to IdUrd treatment were composed of a mixture of the Le and F antigenic types, with the latter forming a minor species. In contrast, no detectable F interferon was present in spontaneously produced IF preparations from Namalwa cells, or after induction with B95-8 virus.
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Expression of the Genome of Defective Interfering Pseudorabies Virions in the Presence or Absence of Helper Functions Provided by Standard Virus
More LessSUMMARYThe synthesis of virus RNA and proteins in cells infected with two populations of defective virions [Pr(1)53 and Pr(2)53] which vary in the overall composition of their DNA, but which share some structural and biological characteristics, have been examined. The experiments were done under two sets of conditions: (1) at high multiplicity of infection. In this case, practically all the cells in the cultures were co-infected with defective and infectious virions; (2) at low multiplicity of infection. In this case, 75% of the cells in the cultures were infected with defective virions only and 25% were co-infected with defective and infectious virions. The relative abundance of RNA classes complementary to different regions of the virus genomes that were synthesized under various conditions of infection were determined by the Southern (1975) technique; the synthesis of virus proteins was determined by polyacrylamide gel electrophoresis (PAGE). In cells co-infected with standard and defective virions, RNA complementary to the regions that are reiterated in the defective genomes is present in larger amounts than in cells infected with standard virions alone, indicating that the genomes of the defective virions are transcribed. Furthermore, the transcriptional controls that operate normally in the infected cells also operate in cells co-infected with standard and defective virions. The over-abundant accumulation of transcripts of some regions of the virus genome in cells co-infected with defective virions is not necessarily accompanied by an overproduction of some virus proteins. No difference in the PAGE profiles of the proteins synthesized was detected in cells co-infected with Pr(1)53 and standard virions. However, cells co-infected with standard and Pr(2)53 overproduced three polypeptides. Transcription of the virus genome is detectable in cells infected with Pr(2)53 alone but not in cells infected with Pr(1)53 alone. Virus protein synthesis is also detectable under these conditions in Pr(2)-, but not in Pr(1)-infected cells. Thus, despite the similarities in the biological characteristics of the two populations of defective virions described previously, similarities with respect to the expression of their genomes were not found.
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Small Virus-like Particles in Honey Bees associated with Chronic Paralysis Virus and with a Previously Undescribed Disease
More LessSUMMARYChronic bee-paralysis virus associate (CPVA) and cloudy wing particle (CWP) are about 17 nm in diam. contain RNA, have single polypeptide species and have indistinguishable buoyant densities in CsCl of about 1.38 g/ml; but they are serologically unrelated, have proteins of different mol. wt. and have significantly different sedimentation coefficients of 41S and 49S respectively. CPVA is significantly and possibly specifically associated with chronic paralysis virus but multiplies, relative to the paralysis virus, by different amounts in different tissues. CWP multiplies apparently independently.
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Persistent Theiler’s Murine Encephalomyelitis Virus Infection in Mice depends on Plaque Size
More LessSUMMARYTheiler’s murine encephalomyelitis virus (TMEV) is an enteric pathogen of mice which causes acute and chronic neurological disorders in the natural host. When brain-derived stocks of TMEV isolates are adapted to cell culture they predominantly form either large or small plaques. In this study the type of central nervous system (CNS) infection (acute versus chronic) and the associated disease occurring in mice inoculated intracerebrally with large and small plaque strains of TMEV was investigated.
Large and small plaque strains of TMEV were found to vary in virulence, type of neurological disease produced and ability to establish persistent CNS infection in mice. Two large plaque strains, GDVII and FA viruses, were highly virulent, produced acute encephalitis, but were cleared from the nervous systems of surviving animals. Therefore, it appears that these large plaque variants do not cause persistent CNS infection in mice.
In contrast, five small plaque strains, DA, WW, TO4, Yale and BeAn8386 viruses, were relatively avirulent, usually produced no illness during the first month after inoculation, but readily established persistent CNS infection in mice. Persistently infected mice later developed demyelinating disease. Having identified strains of TMEV that differ regarding their ability to persist, we now hope to be able to exploit this difference in elucidating the basic mechanism(s) of TMEV persistence.
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Biochemical Aspects of Variation in Foot-and-Mouth Disease Virus
More LessSUMMARYThe biochemical basis for variation in foot-and-mouth disease virus (FMDV) has been explored by analysis of the virus RNA and the virus-induced and structural proteins of three isolates of the virus. Two of the isolates were from serotype A and the third was from serotype O. Hybridization studies of the RNAs showed greater than 80% homology between the two type A viruses and about 65% homology between the two type A viruses and the virus of type O. The ribonuclease T1 maps of the three viruses gave distinct patterns typical of FMDV, but did not show that any two of the three viruses were more closely related. The virus-induced primary translation products, P88, P52 and P100 isolated from infected cells, were compared by tryptic peptide analysis. Combinations of 3H- and 14C-leucine-labelled polypeptides were hydrolysed with trypsin and resolved on an ion-exchange column. Much greater differences were found in P88 than in P52 or P100, indicating that the major variation occurs in the region of the genome coding for the structural proteins. Similar analysis of combinations of the structural proteins of the three viruses showed that there were differences in VP1, VP2 and VP3 and these results were supported by those obtained by PAGE analysis of the Staphylococcus aureus V8 protease cleavage products.
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Inhibition of Moloney Leukaemia Virus Production by N-methylisatin-β-4′:4′-diethylthiosemicarbazone
More LessSUMMARYN-methylisatin-β-4′:4′-diethylthiosemicarbazone (M-IBDET) inhibited the production of Moloney leukaemia virus (MLV). Virus inhibition was related to drug concentrations and time of treatment. The effective antiviral drug concentrations ranged between 3.4 µm and 34 µm. Virus reverse transcriptase activity even at concentrations of 34 µm-M-IBDET was not inhibited. At virus inhibitory concentrations the drug reduced RNA synthesis only very slightly and did not affect protein synthesis at all, although growth and DNA synthesis of host cells were suppressed. The inhibition of cellular DNA synthesis was reversible. Comparison of M-IBDET with actinomycin D, cycloheximide and α-amanitin in terms of their inhibitory effect on the release of MLV into the culture medium showed that M-IBDET was comparable to the other antimetabolites. The inhibition of MLV production by M-IBDET was confirmed by various parameters of virus assay. It was concluded from the experimental evidence that M-IBDET specifically inhibits MLV production.
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Influence of Input Multiplicity of Infection on the Antiviral Activity of Interferon
More LessSUMMARYThe antiviral activity of interferon was shown to be dependent on the input m.o.i. Cells could not be protected against the cytopathogenic effect of vaccinia, herpes, Echo or vesicular stomatitis virus at m.o.i. > 1. At a m.o.i. of ⩽ 1, cells could be protected but the amount of interferon necessary to yield protection was inversely related to the m.o.i. When protection was afforded, it was only transient. The duration of the antiviral effect of interferon was also inversely related to the m.o.i.
The dependence of the antiviral effect on the m.o.i. could not be explained by assuming the viruses to be mixtures of subtypes with different interferon sensitivity. Also, selection by interferon treatment of interferon-insensitive subtypes could not be shown. The greater antiviral effect of interferon at low m.o.i. was probably not caused by induction of interferon by the infecting virus. A direct inactivation by the virus of the antiviral effect of interferon could not be demonstrated. These results indicate that when interferon-treated cells are infected, they will not survive the infection. The only result of the interferon treatment will be to inhibit virus replication to some extent, leading only to a delay in cell death.
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A Plaque Assay for Feline Panleukopenia Virus
More LessSUMMARYPlaque formation with representative strains of feline panleukopenia virus (FPV) has been obtained using a permanent line of feline kidney cells under agarose overlay. FPV-infected cells appear as white plaques after neutral red staining. Plaque size is determined by the extent of cell division in the infected monolayer. FPV assay by the plaque procedure is rapid and gives infectivity titres which exceed those determined by the common inclusion body and immunofluorescent assays of FPV by a factor of about 100 and 10, respectively. Moreover, the plaque assay offers an effective means for the quantification of neutralizing antibodies in feline sera as well as for the detection of heat-stable substances in bovine sera which strongly interfere with replication of the virus.
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Persistent Infection of a Cell Line of Mouse Origin after Cell Fusion by u.v.-inactivated Sendai Virus
More LessSUMMARYA cell line derived from Sendai virus-induced fusion of human adenocarcinoma and CBA mouse embryo cells had Sendai virus antigen (detected by immunofluorescence), together with bi-armed marker chromosomes, in 100% of the cells. After repeated passage, antigen-free cells carrying the same marker chromosomes appeared in the culture. Acrylamide gel analysis showed that all the Sendai virus antigens of antigen-positive cells were normal with the exception of the M protein. Antigen-negative cells contained no virus proteins and could be superinfected with wild-type virus, when all virus proteins appeared.
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Interferon Effects on Friend Leukaemia Cells. I. Expression of Virus and Erythroid Markers in Untreated and Dimethyl Sulphoxide-treated Cells
SUMMARYThe effects of low doses (40 to 1000 units/ml) of mouse interferon (IF) on the expression of Friend leukaemia virus (FLV) and globin genes in Friend leukaemia cells (FLC) have been examined. IF blocks production of extracellular virus, but virus antigens accumulate in the cytoplasm. In cells treated with IF at the time of seeding, there is a reduction in the amount of RNA specified by the lymphatic leukaemia virus (LLV) component of FLV; with the same IF dose there is a small but definite stimulation of haemoglobin and globin mRNA synthesis. The effects of IF on LLV gene expression are even more pronounced in dimethyl sulphoxide (DMSO)-stimulated FLC. No correlation was found between LLV gene expression and the appearance of erythroid markers.
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DNA Synthesis and Multinucleation of Mouse Cells Infected with SV40 in the Presence of Cytochalasin B
More LessSUMMARYDNA synthesis and nuclear division of mouse cells, BALB/3T3, infected with SV40 were studied and were compared with those of SV40-transformed mouse cells, mKS-A TU-7. Both SV40-infected and SV40-transformed cells behaved similarly in the presence of cytochalasin B and differently from normal non-infected cells, BALB/3T3. The chemical inhibited cytokinesis of all the cell groups but the nuclear division was inhibited only in the case of non-infected BALB/3T3. With SV40-infected BALB/3T3, multinucleation occurred with the increase of input m.o.i. by SV40. SV40-infected BALB/3T3 could enter the second S phase after release from double thymidine block in the presence of cytochalasin B, while BALB/3T3 could not enter the second S phase. In bi- or multinucleated cells of SV40-infected BALB/3T3, asynchrony of DNA synthesis among nuclei in a cell was evident, as was the case with mKS-A TU-7.
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Production and Initial Characterization of Rat Interferon
More LessSUMMARYRat interferon of relatively high specific activity (about 106 units/mg protein) was produced in embryonic rat cells treated with Newcastle disease virus at a high m.o.i. The cells were cultured in serum-free medium and the interferon was precipitated and concentrated with 0.02 m-zinc acetate or with ammonium sulphate at 85% saturation. With both methods the increase in interferon activity was greater than the concentration factor. The rat interferon activity was stable on treatment with 0.15 m-perchloric acid or three cycles of freezing and thawing, but incubation at 37 °C for 1 h resulted in a 50% loss in activity. It had no cross activity in human or mouse cells. The sensitivity of different types of rat cells for rat interferon differed widely and was dependent on the challenge virus. Human interferons had no detectable antiviral activity on rat cells and did not block the activity of rat interferon.
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Morphology of a Reo-like Virus Isolated from Juvenile American Oysters (Crassostrea virginica)
T. R. Meyers and K. HiraiSUMMARYRotational enhancement of image detail performed on a proposed new serotype of reovirus suggested icosahedral symmetry of T = 3 with some morphological features of members of the Reoviridae family.
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- Plant
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Tobacco Mosaic Virus Infection Does Not Alter the Polyadenylated Messenger RNA Content of Tobacco Leaves
More LessSUMMARYMeasurements of incorporation of 3H-histidine into proteins in discs from tobacco mosaic virus-infected leaves suggest that the rate of host protein synthesis is reduced by up to 75% during virus multiplication but then recovers. Polyadenylated messenger RNAs from healthy and virus-infected plants were found to have similar size distributions and polyadenylic acid chains of similar lengths. Tobacco mosaic virus infection did not cause any alteration in the concentration of host polyadenylated messenger RNA. This makes it unlikely that rates of transcription or turnover of host polyadenylated messenger RNA were altered by infection. It is suggested that inhibition of host protein synthesis during virus multiplication may result from controls at the translational level, possibly by competition between the messenger RNAs for virus and host proteins.
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Viroid-like Properties of an RNA Species Associated with the Sunblotch Disease of Avocados
More LessSUMMARYA low mol. wt. RNA species is associated with sunblotch disease of avocados. The RNA (SB-RNA) was recovered from leaves and bark of symptomless carrier trees and from bark lesions of symptom-bearing trees. It was not detected in leaves or bark from lesion-free areas of symptom-bearing trees or in leaf or bark from healthy trees.
SB-RNA is soluble in LiCl and migrates in 20% polyacrylamide gels. In high salt buffers, it is resistant to ribonuclease A at a concentration of 1 µg/ml and is degraded only slowly at 10 µg/ml. The RNA is less resistant to ribonuclease A at low salt concentrations (0.01 m). The mobility of the SB-RNA in polyacrylamide gels is not affected by heat denaturation. The apparent mol. wt. of native SB-RNA is 65000 whilst under denaturing conditions it is 112000 to 115000.
SB-RNA occurs in high concentration in leaves of symptomless carrier trees being detected in 1 g of fresh leaf. In tissue fractionation experiments, SB-RNA was associated mainly with the chloroplast and endoplasmic reticulum fractions. It is concluded from these properties of SB-RNA that sunblotch is a viroid disease.
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