CsCl Density Research Articles

RNA-Friendly Plasmid Preparation

With the advent of miniprep kits for the preparation of plasmids, we have noticed that many investigators use plasmids prepared by these methods as transcription templates or for cell transfection experiments. This can be quite problematic because miniprep (or maxiprep) DNA can often be contaminated with RNases. Indeed, most DNA preparation kits include pancreatic RNase. This RNase is extremely difficult to inactivate and can contaminate benchtops and pipettors. We highly recommend that plasmid DNAs, especially those that will be used repeatedly, be purified using traditional cesium chloride (CsCl) density gradient methods that do not involve the use of RNase. We have found that the protocol described here yields highly pure, noncontaminated plasmid DNA suitable for in vitro transcription and/or transfection analyses. An added benefit to this procedure is that it will yield enough plasmid DNA for many, many experiments.

Human CST Has Independent Functions during Telomere Duplex Replication and C-Strand Fill-In

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here, we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a replication factor that solves diverse replication-associated problems.

Diversity of Flavobacterium psychrophilum and the potential use of its phages for protection against bacterial cold water disease in salmonids

Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.

New tombusviruses isolated from surface waters in New Zealand

Water samples from Turitea Creek and the Manawatu River near Palmerston North, New Zealand were assayed for infectious plant viruses. Twenty liter water samples each were prefiltered and virions adsorbed onto electropositive Zeta Plus 50S membranes. Eluates were examined for virions by transmission electron microscopy. Icosahedral particles with a diameter of 30 nm with no distinct capsomere arrangement were observed. Two distinct tombusvirus isolates were suspected based on symptoms in Vigna unguiculata. An A260/280 = 1.64, and a buoyant density in CsCl = 1.35 supported the conclusion that both isolates were tombusviruses. Maximum Parsimony trees generated from the deduced amino acid sequence of an 820 bp amplicon within the p33 (RdRp) gene and the entire capsid protein gene (1100 bp) showed maximum similarity of the isolates from Manawatu river and Turitea creek with Cucumber bulgarian latent virus (60% and 64%, respectively) in the p33 region, and with Maize necrotic streak virus (83%) and Cymbidium ringspot virus (76%), respectively, in the capsid protein region. In accordance with the species demarcation criteria of <87% sequence identity in the capsid protein amino acid sequences established by the ICTV, it is suggested that both isolates are new tombusviruses, for which we propose the names Turitea creek virus (TuCV) and Manawatu river virus (ManRV). This is the first report of tombusviruses in New Zealand.

Bromodeoxyuridine-labeled bacteriophage T4D. I. Growth parameters and frequency of markers recovered from BUdR-labeled and from non-labeled phages studied in mass lysates.

T4D bacteriophages, grown on heavy Escherichia coli strain CR63 in a synthetic medium containing BUdR, FUdR and uracil, showed a uniform BUdR labeling as judged from visible light inactivation and from CsCl density gradients. A variable number of “dead” BUdR phages were found in different BUdR phage lysates by comparing plaque former titer with complementation titer. The “dead” BUdR phages had the same mean density in CsCl as plaque-forming BUdR phages from the same lysate, indicating that “dead” BUdR phages do not suffer from a large deficiency of DNA. The “dead” BUdR phages participated in multiplicity reactivation. Production of intracellular phages was delayed in BUdR experiments compared to control experiments, especially when both parents in a cross were BUdR labeled, and when BUdR phages were used for single infection. In all Type A crosses (one parent BUdR-labeled, one parent non-labeled) the recovery of markers was significantly lower from BUdR parents than from the corresponding non-labeled parents in the control crosses. In one Type B experiment (both parents BUdR-labeled) the frequency of recombinants was significantly higher than in all Type A experiments.

High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.

Genomic characterization of the unclassified bovine enteric virus Newbury agent-1 (Newbury1) endorses a new genus in the family Caliciviridae

The pathogenic bovine enteric virus, Newbury agent-1 (Bo//Newbury1/1976/UK), first identified in 1976, was characterized as a possible calicivirus by morphology, buoyant density in CsCl and the presence of a single capsid protein but genomic sequence could not be obtained. In the present study, the complete genome sequence of Newbury1 was determined and classified Newbury1 in a new genus of the Caliciviridae. The Newbury1 genome, of 7454 nucleotides, had two predicted open reading frames (ORFs). ORF1 encoded the non-structural and contiguous capsid proteins. ORF2 encoded a basic protein characteristic of the family Caliciviridae. Compared to the 4 recognized Caliciviridae genera, Norovirus, Sapovirus, Lagovirus and Vesivirus, Newbury1 had less than 39% amino acid (47% nucleotide) identity in the complete 2C-helicase, 3C-protease, 3D-polymerase and capsid regions but had 89% to 98% amino acid (78% to 92% nucleotide) identity to the recently characterized NB virus in these regions. By phylogenetic analyses, Newbury1 and NB viruses formed a distinct clade independent of the 4 recognized genera. However, amino acid identities showed that Newbury1 and the NB virus were distinct polymerase types (90% amino acid identity), but their complete capsid proteins were almost identical (98% amino acid identity). Analyses of contemporary viruses showed that the two polymerase genotypes, Newbury1 and NB, were circulating in UK cattle and antibody to Newbury1-like viruses was common in cattle sera. The present study defined the existence of a new genus in the Caliciviridae that we propose be named Becovirus or Nabovirus to distinguish the new clade from bovine noroviruses.

Expression of hepatitis B surface antigen in transgenic banana plants

Embryogenic cells of bananan cv. Rasthali (AAB) have been transformed with the 's' gene of hepatitis B surface antigen (HBsAg) using Agrobacterium mediated transformation. Four different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilized to optimize the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen was confirmed by PCR, Southern hybridization and reverse transcription (RT)-PCR. The expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by ELISA for all the four constructs. Maximum expression level of 38 ng/g F.W. of leaves was noted in plants transformed with pEFEHBS grown under in vitro conditions, whereas pHER transformed plants grown in the green house showed the maximum expression level of 19.92 ng/g F.W. of leaves. Higher monoclonal antibody binding of 67.87% of the antigen was observed when it was expressed with a C-terminal ER retention signal. The buoyant density in CsCl of HBsAg derived from transgenic banana leaves was determined and found to be 1.146 g/ml. HBsAg obtained from transgenic banana plants is similar to human serum derived one in buoyant density properties. The transgenic plants were grown up to maturity in the green house and the expression of HBsAg in the fruits was confirmed by RT-PCR. These transgenic plants were multiplied under in vitro using floral apex cultures. Attempts were also made to enhance the expression of HBsAg in the leaves of transgenic banana plants by wounding and/or treatment with plant growth regulators. This is the first report on the expression of HBsAg in transgenic banana fruits.

Profiles of HCV core protein and viremia in chronic Hepatitis C: possible protective role of core antigen in liver damage

The relation between HCV core antigen and HCV RNA has been confirmed in patients with chronic hepatitis C and a parallel behavior of the two markers has been described in early kinetics analysis during antiviral therapy. Variations of the core antigen to HCV RNA ratio have been reported in individual patients and the existence of nucleocapsid particles, not always associated with viral genomes, have been described. To assess the characteristics of HCV core antigen reactivity in relation to viremia in patients with different clinical profiles, 233 patients with chronic hepatitis C were studied serially. Group A included 54 asymptomatic HCV carriers, group B included 8 viremic patients with biochemical long-term response after antiviral therapy, while group C was composed of 171 patients with chronic liver disease and 75 were treated with combination therapy. Core antigen levels were not significantly different in the three groups of patients and a wide range of antigenic reactivity was observed in individual patients. A close relationship was observed between core antigen and HCV RNA, although their ratio was significantly higher in biochemical long-term responders (group B), compared to the other groups (P < 0.05). Physicochemical characterization of core antigen reactivity by equilibrium CsCl density gradient identified two distinct peaks migrating at 1.08-1.12 g/ml CsCl density and at 1.18-1.31 CsCl density, respectively. The first one, corresponding to the lipid-associated fraction, contained higher amounts of core antigen reactivity and was associated with clinical remission of liver damage, while the second peak, corresponding to naked nucleocapsids, was observed mainly in sera with active disease. In conclusion, a close relationship between core and HCV RNA was documented both in treated and untreated patients. The finding of an excess of lipid-associated core particles in a subset of viremic patients without biochemical activity of liver disease suggests their protective effect in liver cell damage.

Expression of hepatitis B surface antigen in tobacco cell suspension cultures

Hepatitis B virus ‘ s’ gene coding for surface antigen was cloned into plant transformation vectors pHER100 and pHBs100 with and without endoplasmic reticulum retention signal, respectively. Transformed tobacco cell lines were analyzed for the integration of the transgene by PCR and Southern blot hybridization. Expression levels as determined by ELISA showed maximum expression levels of 2 μg HBsAg gm −1 fresh weight and 10 ng mL −1 of spent medium in pHER100 transformed cells. Western blot analysis confirmed the presence of 24 kDa band specific to HBsAg in the transformed cells. HBsAg was expressed both as intracellular and secreted forms in pHER100 transformed cells. The buoyant density in CsCl of HBsAg derived from pHBs100 transformed tobacco cells was determined and found to be 1.095 g mL −1. HBsAg obtained from transformed tobacco cells is similar to the human serum derived one in buoyant density properties. This is the first report on the secretion of HBsAg particles by plant cells into the cell culture medium.

Characterization of mycoviruses and analyses of chitinase secretion in the biocontrol fungus Metarhizium anisopliae.

Metarhizium anisopliae is the best-characterized entomopathogen and is used to control insect pests in sugar cane plantations in Brazil on a commercial scale. We have previously reported the infection of some M. anisopliae strains by dsRNA mycoviruses. Here we describe the purification and characterization of the viruses (MaV-A1, MaV-M5, MaV-RJ) in terms of dsRNA content, capsid proteins, electron microscopy, Western blot, and hybridization patterns. One spontaneous mutant lost some of the high molecular weight dsRNA components and showed significant alterations in colony morphology and spore production, suggesting that viral genes interfere with fungal phenotype. A comparison between dsRNA mycovirus-free and infected M. anisopliae isolates showed that virus-free isolates have increased endochitinase secretion. By comparing the following parameters: the buoyant density in CsCl of the presumed virions; the number and estimated molecular weight of the dsRNA components and the molecular mass of the capsid proteins to other mycoviruses previously described, we suggest the inclusion of MaV-A1 and MaV-M5 in the family Totiviridae and MaV-RJ in the family Partitiviridae.

Purification of DNA for the transfection of a Spodoptera frugiperda cell line.

Spodoptera frugiperda (Sf-9) cells have been widely used in baculovirus expression systems, transient gene expression studies and transgenic cell lines. These applications commonly require the transfection of bacterial plasmid DNA. One of the most reliable methods of preparing transfection-quality plasmid DNA is cesium chloride (CsCl) density gradient centrifugation. However, the traditional CsCl DNA purification is a long and laborious process. We have made a series of modifications to the traditional method that makes it faster, safer and easier. In the current study we demonstrate that DNA prepared by our modified CsCl method was also better for the transfection of Sf-9 cells than DNA prepared by the traditional CsCl method.

Characterization of a Virus from Pigeonpea with Affinities to Species in the Genus Aureusvirus, Family Tombusviridae.

In attempts to identify the causal agent of pigeonpea sterility mosaic disease (PSMD), which is transmitted by eriophyid mites, a virus was isolated with great difficulty from some PSMD-affected pigeonpea (Cajanus cajan) plants from different locations in India. Once isolated from pigeonpea, the virus was transmitted readily by mechanical inoculation to several herbaceous species, reaching very high concentrations in some species. The virus was transmitted experimentally through soil to herbaceous test plants but not to pigeonpea. When virus particles were purified and inoculated mechanically to healthy pigeonpea, the virus induced necrosis in inoculated leaves only and did not spread systemically. Therefore, the virus is not the causal agent of PSMD. The virus has isometric particles approximately 30 nm in diameter that sediment as a single component and had a buoyant density in CsCl and Cs2SO4 of 1.34 and 1.27 g·cc-1, respectively. Purified virus particle preparations contained a single major protein of approximately 44 kDa and three RNA species of approximately 4,300, 2,700, and 1,500 nucleotides. Only the largest RNA species was infective to plants; the two smaller species were encapsidated subgenomic species of the 3' end of the larger genomic RNA. The viral genome was sequenced and showed 93% homology to that of Pothos latent virus (PoLV), a recently described virus in the genus Aureusvirus, family Tombusviridae, and was indistinguishable from PoLV in gel double-diffusion serological tests. This virus, therefore, is regarded as a pigeonpea isolate of PoLV (PoLV-PP). In field studies in different locations in India, enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction detected PoLV-PP in 10.7% of PSMD-affected and 8.1% of asymptomatic pigeonpea plants. The significance of these findings is discussed.

Detection and Partial Characterization of Tenuiviruses from Black Spruce.

Filamentous viral ribonucleoproteins (RNPs) 12 to 16 nm in diameter and 100 to 1,260 nm in length, and characteristic of the genus Tenuivirus, were detected by transmission electron microscopy in purified extracts of needles collected from two mature, asymptomatic black spruce (Picea mariana) trees in New York, but not in extracts of needles from nursery seedlings. Purified RNPs from one tree had a buoyant density in CsCl = 1.39 g/cm3 and an A 260/280 = 1.436. Four ssRNA segments of 1.3, 2.1, 2.3, and 3.5 kb, but not the 8- to 9-kb fragment characteristic of most tenuiviruses, were detected in purified RNA extracts. Amplification products of the expected size were observed when RNA extracts from the two spruce trees and Maize stripe tenuivirus (MStpV), but not from tobacco, Chenopodium quinoa, or spruce seedlings were subjected to reverse transcription-polymerase chain reaction (RT-PCR) using primers to the p3 open reading frame (ORF) of MStpV vRNA 3. However, only MStpV amplified when primers to the nucleocapsid ORF (pc3 ORF on vcRNA 3) were used. Similarly, only MStpV amplified by immunocapture polymerase chain reaction (PCR) using antiserum to MStpV and primers to the p3 ORF. Sequence comparisons suggest that two distinct tenuiviruses occur in black spruce, one more closely related to MStpV than the other. One of these tenuiviruses was detected in one of 10 additional black spruce trees tested, but not in trees of six other coniferous species sampled in the Adirondack Mountains of New York.

Heterogeneous response for a mammalian hepadnavirus infection to acyclovir: Drug-arrested intermediates of minus-strand viral DNA synthesis are enveloped and secreted from infected cells as virion-like particles

Three woodchucks infected persistently with the woodchuck hepatitis virus (WHV) were treated with acyclovir (ACV) to investigate the effect of inhibiting viral DNA synthesis upon the replication of an orthohepadnavirus in vivo. Normal viraemia was reduced during the treatment period in all three animals, but each responded with a distinct serum phenotype. In the most provocative case, the profile of the WHV DNAs in both the liver and serum provided a simple and novel description of the orthohepadnaviral infection for this ACV protocol. The pre-drug viraemia was rapidly cleared from the serum and replaced by virion-like particles containing predominantly minus-strand WHV DNAs. These serum DNA species had the character of replicative intermediates arrested in their elongation by ACV-mediated chain termination and were contained in particles with a buoyant density in CsCl essentially identical with virions. However, in infected hepatocytes, initiation of reverse transcription within newly formed core particles was not inhibited by the ACV treatment. Instead, an heterogeneous array of minus-strand DNAs were synthesised, each presumed to be truncated by the incorporation of one molecule of ACV monophosphate. An approximately normal level of core particles was present in the liver of this woodchuck after 26 days of the ACV protocol; excess drug-arrested nucleocapsids were steadily removed throughout the dosing period upon their envelopment and secretion as virion-like particles into the circulation. These data suggest that plus-strand DNA synthesis may not be absolutely required prior to secretion of virus from the infected cell.

Characterization of chilli vein‐banding mottle virus isolated from pepper in Thailand

A virus with flexuous particles, previously designated chilli veinal mottle virus (CVMV)‐CM1, which causes dark green spot, green vein‐banding and leaf distortion on peppers in Thailand, was isolated and characterized. The host range of this virus was restricted to the Solanaceae. The mean particle length in crude sap was 765 nm and the mean particle width was 13 nm. The virus was purified from pepper or Nicotiana glutinosa by extraction in phosphate buffer, followed by chloroform clarification and precipitation with PEG. The partially purified virus was further purified by sucrose density gradient centrifugation. This protocol consistently yielded 8–19mg virus/kg infected leaves. The purified virus had a UV absorption spectrum typical of nucleoprotein, with a minimum at 246 nm, a maximum at 260 nm, and a slight shoulder at 290 nm. The A260/A280 ratio was in the range 1·20–1·28, and the Amax/Amin ratio was in the range 1·11–1·27; the buoyant density in CsCl was 1·328g/cm3 Virions contained a single‐stranded RNA of c. 10kb and a single major protein of molecular weight 34000. The virus induced cytoplasmic inclusions in infected cells, which were observed as pinwheels, scrolls and laminated aggregates. It was not serologically related to potato virus Y (PVY, PVY‐O, PVY‐T), pepper mottle virus (PepMoV), pepper veinal mottle virus (PVMV), tobacco etch virus (TEV), papaya ringspot virus (PRSV), or peanut stripe virus (PStV), but is related to chilli veinal mottle virus occurring in Malaysia. On the basis of particle morphology, physico‐chemical and biological properties and cytopathology, it is considered to be a potyvirus. As this virus was serologically distinct from well‐known potyviruses, it is thought to be a new pepper potyvirus found in Thailand. Since the virus induces a typical dark green spotting, patching and banding along the vein in established infected peppers, the new and more descriptive name ‘chilli vein‐banding mottle virus’(CVbMV) is proposed.

Characterization of 45S ribonucleoprotein particles detected in wheat embryo cytoplasm

Relatively homogeneous 45S ribonucleoprotein particles containing considerable amounts of newly-synthesized rapidly labelled RNA were detected in cytoplasmic extract from germinating wheat embryos. These particles have the buoyant density in CsCl of about 1.50 g/cm 3 and apparently are not represented by free cytoplasmic informosomes. The isolated particles contain 17S rRNA, rapidly-labeled heterogeneous (6–16S) RNA with strong messenger activity and several low molecular weight RNAs among which the 5.3S RNA (of about 135 nt) is the most abundant. According to 2D-PAGE two groups of polypeptides can be revealed in the particles. Polypeptides of the first group (10–45 kDa) correspond to structural ribosomal proteins. The second group consists of higher molecular weight polypeptides (30–100 kDa) and may include polypeptides of mRNP and translation initiation factors. The 45S particles may represent a native [mRNP × 40S RS × eIFs × Met-tRNA i × GTP] complexes accumulating in vivo as a translation initiation intermediate.

The General Characteristics of a Birnavirus Isolated from Cultured Loach(Misgurnus anguillicaudatus) in Taiwan.

A birnavirus was isolated from cultured loach (Misgurnus anguillicaudatus) in Taiwan with TO-2 cell line. Evident cytopathic effects of the virus, including karyopycnosis, cytoplasmic inclusion bodies and cell destruction, were observed in TO-2 cells by light microscopy. The naked icosahedral virion layered by a single capsid with a diameter of 69 ± 4 nm was observed by electron microscopy. Sometimes, complete virions were found to be enclosed by lysosomes to form cytolysome. The virus propagated in several fish cell lines and its replication cycle was completed in 24 hours at its optimum multiplication temperature of 25°C. This virus has a two segmented double-stranded RNA genome and 3 structural proteins, showing 1.33 g/cm3 buoyant density in CsCl. The chemico-physical properties of the virus together with the result of cross neutralization test indicated that it is a birnavirus and is closely related to Ab serotype of infectious pancreatic necrosis virus (IPNV).

Two novel viruses associated with severe disease symptoms of the green stinkbug Nezara viridula.

Two viruses were isolated from green stinkbugs (Nezara viridula) with severe disease symptoms. These viruses have been named N. viridula virus type 1 (NVV-1) and NVV-2 according to their relative sedimentation coefficients. NVV-1 is a small picorna-like virus with a diameter of 29 nm, a buoyant density in CsCl of 1.34 g/ml and a sedimentation coefficient of 153S. NVV-1 particles contain a 9.4 kb ssRNA segment and have three coat proteins of M(r)s 32,100, 31,500 and 30,700. NVV-2 sediments as two components on sucrose gradients; the top 104S component consists almost entirely of 41 nm empty capsids and the faster sedimenting 177S component consists of intact 39 nm spherical particles. NVV-2 particles have a buoyant density in CsCl of 1.39 g/ml and consist of one major protein of M(r) 73,800 and at least two minor proteins of M(r)s 13,500 and 16,500. Only one dsRNA segment of 6.2 kb was identified. The properties of NVV-2 are similar to those of the Totiviridae. Individual stinkbugs were infected with either NVV-1 or NVV-2, or with a mixture of the two viruses. Re-infection of virus-free stinkbugs with the mixture resulted in typical disease symptoms. Both viruses were vertically transmitted through the eggs and insects were infected by surface contamination of their food source.

Purification and Properties of Spherical Virus Particles Associated with Grapevine Ajinashika Disease

Grapevine ajinashika associated virus (GAaV) was successfully purified from ajinashika-affected berry pulps by an enzyme treatment purification procedure. Virions were spherical (approximately 25 nm in diameter), sedimented as a single component of approximately 110S, had a buoyant density in CsCl of approximately 1.38 g/cm 3 , and had an estimated nucleic acid content of 30%. GAaV contained a single species of single-stranded RNA of an estimated 6.8 kb and a coat protein of 23,000 Da