CsCl Isopycnic Centrifugation Research Articles

Cloning and characterization of an equine cutaneous papillomavirus

Equine papillomaviruses (EgPV) from naturally occurring cases of cutaneous papillomatosis in several ponies and one horse were isolated, cloned, and characterized. Group specific papillomavirus structural antigens were detected in sections of the papillomas by the peroxidase-antiperoxidase technique, and virions were observed in the nuclei of cells in the stratum granulosum and corneum. Negatively stained virions purified from papilloma homogenates by isopycnic CsCl centrifugation were 55 nm in diameter and had typical papillomavirus morphology. The entire viral genomes of two separate isolates were cloned at a single BamHl site into pBR322. A detailed restriction map of the viral genome is presented. Using nick-translated subgenomic fragments of BPV-1 as probes in Southern blot hybridizations, the organization of the EgPV genome was established. Southern blot analysis under various conditions of stringency revealed that EgPV shares relatively more homology with the late region of the BPV-1 genome and with the E2 region of the HPV-1 genome than with other parts of the same viral DNAs. Papillomavirus-specific sequences were found in papillomas from other anatomic sites using the EgPV DNA as a probe in Southern blot hybridizations. Genomes detected in DNA from penile papillomas had a different restriction pattern and hybridized to the EgPV probe only under nonstringent conditions.

Organization of glycosaminoglycan chains in a chondroitin sulfate - dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate - dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by β-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.

Melon rugose mosaic virus, the cause of a disease of watermelon and sweet melon

SUMMARYA mechanically transmissible virus with isometric particles c. 32 nm in diameter, was isolated from infected watermelons and sweet melons in the People's Democratic Republic of Yemen. Purified virus preparations contained two major sedimenting components with sedimentation coefficients of 61S and 117S. In isopycn ic centrifugation in CsCl the particles formed a single band of buoyant density 1.39 g cm‐3. Preparations of virus particles comprised of a single polypeptide of mol. wt c. 22 000 and ssRNA of mol. wt 2.1 × 106. The virus was serologically related to three of six subgroups of tymoviruses tested. The name melon rugose mosaic virus is proposed for this newly described virus.

Three seedborne cryptic viruses containing double-stranded RNA isolated from white clover

White clover cryptic viruses (WCCV) 1, 2, and 3 were purified from apparently healthy plants with yields of about 200 μg of virus/kg of tissue. After isopycnic centrifugation in CsCl, WCCV 1 and 2 formed homogeneous bands at densities of 1.392 and 1.375 g/ml, respectively, whereas WCCV 3 was not recovered. Particles of WCCV 1 and 3 were 34 nm in diameter, without obvious morphological subunits, whereas WCCV 2 particles were 38 nm in diameter, with prominent morphological subunits. Antisera with titers up to 1:2048 were obtained against WCCV 1 and 2. Each of the three viruses contained two segments of linear dsRNA ( M r from 1.03 to 1.49 X 10 −6. Virus-like particles, probably those of WCCV 1 and 2 were detected in low numbers in the cytoplasm of about 1/10 of the parenchyma cells of carrier plants examined by thin sectioning. The particles of WCCV 1 and 2 were not transmitted by grafting or mechanical inoculation, but were seed transmitted. Evidence is given that WCCV 1 and 2 multiply in their carrier plants, and are not viruses of associated parasitic or commensal organisms.

Attachment of the 5'-terminal portion of globin mRNAs to 5S-RNA X L5-protein in the 80S initiation complex.

An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA X L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. Hybridization experiments, using restriction fragments of cDNA for rabbit beta globin mRNA, showed that the attached mRNA fragments were derived from the 5' portion of globin mRNAs. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5' portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed.

Proteoglycans from lungs of rabbits treated with pronase and cadmium chloride.

The composition of proteoglycans was studied in lungs from rabbits treated intratracheally with pronase, CdCl2, or saline. Proteoglycans were extracted from the lungs by 0.5 M and 4.0 M guanidine hydrochloride (GdnHCl) in sequence. Proteoglycans were isolated from the extracts by CsCl isopycnic centrifugation and fractionated by gel filtration on a Sepharose CL-4B column. The lungs from pronase-treated rabbits had approximately 33% greater total uronate than did lungs from saline-treated control animals, whereas CdCl2-treated rabbit lungs had 28% less uronate than did control lungs. Pronase-treated animal lungs had a greater proportion of hyaluronic acid and dermatan sulfate than did the control animal lungs. Relatively greater amounts of uronate were extracted by 0.5 M GdnHCl from the enzyme-treated rabbit lungs than from the CdCl2- or saline-treated animal lungs. The concentrations of hyaluronic acid and heparan sulfate in 0.5 M GdnHCl extract and dermatan sulfate in 4.0 M GdnHCl extract were greater in pronase-treated animal lungs than in control lungs. Gel filtration of proteoglycans sedimented at q greater than 1.45 revealed that pronase-treated animal lungs had proteoglycans smaller in molecular size than those in control lungs. The observations indicate that intratracheal treatment with pronase and CdCl2 alters the composition of proteoglycans in the lung, and such alterations are likely important in the pathogenesis of emphysema.

Isolation and characterization of a link protein from bovine aorta proteoglycan aggregate

Proteoglycan aggregates were isolated from bovine aorta by extraction with 0.5 M guanidine hydrochloride in the presence of proteinase inhibitors and purified by isopycnic CsCl centrifugation. The bottom two-fifths (A1) of the gradient contained 30% of proteoglycans in the aggregated form. The aggregate had 14.8% protein and 20.4% hexuronic acid with hyaluronic acid, dermatan sulfate and chondroitin sulfates in a proportion of 18:18:69. A link protein-containing fraction was isolated from the bottom two-fifths by dissociative CsCl isopycnic centrifugation. The link protein that floated to the top one-fifth of the gradient was purified by chromatography on Sephadex G-200 in the presence of 4 M guanidine hydrochloride. It moved as a single band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 49 000. The amino acid composition of link protein resembled that of link protein from cartilage, but was strikingly different from that of the protein core of the proteoglycan monomer. The neutral sugar content of link protein was 3.5% of dry weight. Galactose, mannose and fucose constituted 21, 62 and 16%, respectively of the total neutral sugars. In aggregation studies the link protein was found to interact with both proteoglycan monomer and hyaluronic acid. Oligosaccharides derived from hyaluronic acid decreased the viscosity of link protein-free aggregates of proteoglycan and hyaluronic acid but not of link-stabilized aggregates, demonstrating that the link protein increases the stability of proteoglycan aggregates.

The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated.

Mitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by Phenyl-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a protease-insensitive, submolecular domain (Mr approximately equal to 6,000) that retained the capacity to bind tenaciously to the DNA. Analysis of chemically fixed complexes by CsCl isopycnic gradient centrifugation showed that P16 was bound to a large subpopulation of mtDNA enriched in displacement loops (D-loops). Based upon the effective buoyant density of the complex in CsCl gradients and the molecular weights of P16 and mtDNA, it was estimated that a mean of 49 P16 molecules were bound per mtDNA. For this measurement, the variation in hydration of protein and DNA at different CsCl concentrations was ignored. Analysis of restriction endonuclease-digested complexes by glass fiber filters that bind only protein-associated DNA resulted in the retention of a single fragment regardless of the enzyme, or enzymes, used. In each case, the retained fragment was the D-loop-containing fragment. With direct electron microscopy, the protein was readily visualized on the displaced single strand portions of D-loops and expanding D-loops. The nucleoprotein fibers were approximately 12 nm in diameter without correcting for the thickness of tungsten coating and roughly 1/3 the length of the double strand segment of the corresponding D-loop structure. In addition, occasional molecules with the characteristics of gapped circles were seen exhibiting a nucleoprotein fibril, presumably containing the single strand gap segment, linking the ends of double strand DNA. P16 was not seen on the double strand portions in any of the complexes.

Effect of alkyltins on rabbit articular and growth-plate chondrocytes in monolayer culture.

The effect of four different alkyltins (trimethyltin, triethyltin, dibutyltin, and dioctyltin) on the metabolism of rabbit articular and growth-plate chondrocytes was investigated using a monolayer cell-culture system. In most instances the compounds tested exhibited a general cytotoxic effect on these cells, inhibiting the synthesis of both DNA and sulfated proteoglycans. The effect of these compounds on proteoglycan synthesis was both quantitative and qualitative, as demonstrated by CsCl isopycnic density gradient centrifugation and gel exclusion chromatographic techniques. However, certain tin compounds tested, at specific concentrations, exerted a stimulatory effect on chondrocyte proliferation. Regarding DNA synthesis, growth-plate chondrocytes were more sensitive to the effect of the triethyltin, dibutyltin, and dioctyltin than were articular chondrocytes. The data are discussed in relation to the possible effects of the alkyltins on skeletal growth and development as well as the mechanism of action of the alkyltins at the molecular level.

Heterogeneity of heparan sulfate chains in a proteoglycan from bovine lung.

A heparan sulfate proteoglycan from bovine lung gas-exchange tissue was isolated by extraction of the tissue with 4.0 M guanidine HCl in the presence of multiple protein inhibitors. The proteoglycan was purified by precipitation with cetylpyridinium chloride in 0.5 M KCl followed by CsCl isopycnic centrifugation (po = 1.45) in 4.0 M guanidine/HCl. Further purification was achieved by gel filtration on Sepharose CL-2B and by chromatography in DEAE-Sepharose CL-6B column. The proteoglycan had 14.9% protein and 22.4% uronate. Heparan sulfate chains from the proteoglycan were isolated after beta-elimination. Fractionation of heparan sulfate chains was achieved on Dowex-1 Cl- column, eluting with a stepwise increase in the concentration of NaCl, 1.0 to 2.0 M with 0.2 M increments. Of the total heparan sulfate recovered from the column, about 10% eluted by 1.2 M NaCl, 68% by 1.4 M NaCl, 18% by 1.6 M NaCl and 4% by 1.8 M NaCl. The fractions varied in their total and N-sulfate ester contents and iduronic acid to glucuronic acid ratios. The fraction that eluted from the Dowex-1 Cl- column at 1.6 M NaCl had the highest molecular weight, 37000, and the fraction that eluted at 1.8 M NaCl had the lowest molecular weight, 12000, as determined by gel filtration method, and the greatest sulfate content. The core protein, obtained by digestion of proteoglycan by heparan sulfate lyase, showed mostly a single band in SDS-polyacrylamide gel electrophoresis. The observations indicate a heterogeneity of the composition of heparan sulfate chains in the proteoglycan. This heterogeneity likely contributes to variations in biologic properties of different heparan sulfate proteoglycan preparations.

Improved methods for the purification and detection of cocoa swollen shoot virus

SUMMARYThe extraction of cocoa swollen shoot virus (CSSV) from cocoa leaves with pectinase, purification from the concentrated extracts by filtration through Celite and Sepharose 2B, and concentration of the virus by isopycnic CsCl centrifugation are described. The pectinase and Celite treatments effectively removed mucilage and particulate host plant materials, and enhanced the release of virus particles, Isopycnic CsCl centrifugation resulted in 10–100‐fold concentration of particles, but with apparent loss of infectivity.After extraction with pectinase, CSSV particles were regularly seen by electron microscopy in sap. Immunosorbent electron microscopy (ISEM) considerably enhanced the detection of CSSV in sap. The particles were bacilliform or bullet‐shaped and of various lengths. The common dimensions were 142 × 27 nm.

Hemostatic properties and serum lipoprotein binding of a heparan sulfate proteoglycan from bovine aorta

The biologic properties of two major proteoglycans of bovine aorta, heparan sulfate proteoglycan and chondroitin sulfate-dermatan sulfate proteoglycan were compared. The heparan sulfate proteoglycan was isolated either by elastase digestion or by 4.0 M guanidine hydrochloride extraction, of aorta tissue, fractionated by CsCl isopycnic centrifugation and purified by chondrointinase ABC treatment. The first method resulted in considerably greater yield (about 70% of the total heparan sulfate proteoglycan of the tissue) than the second procedure (12% of total). The chondroitin sulfate-dermatan sulfate proteoglycan was obtained by 4.0 M guanidine-HCl extraction of aorta tissue followed by CsCl isopycnic centrifugation. The chemical composition of both heparan sulfate proteoglycan preparations was similar. Unlike the chondroitin sulfate-dermatan sulfate proteoglycan, which eluted in the void volume of Sepharose CL-6B column, the heparan sulfate proteoglycan preparations were each resolved into a high molecular weight fraction ( k av = 0.18 and 0.13) and a low molecular weight fraction ( k av = 0.47 and 0.36). The heparan sulfate proteoglycan preparations exhibited significantly more potent anticoagulant and platelet aggregation inhibitory activities than the chondroitin sulfate-dermatan sulfate proteoglycan. The protein core of the proteoglycan molecules did not seem to be essential for their hemostatic properties. The complex forming ability of the heparan sulfate proteoglycan with serum low density lipoproteins (LDL) was much less than that of chondroitin sulfate-dermatan sulfate proteoglycan in the presence and absence of Ca 2+. Interaction between heparan sulfate proteoglycan and LDL was also much more sensitive to changes in the ionic strength of the medium than that of chondroitin sulfate-dermatan sulfate proteoglycan and the lipoprotein. Since the total sulfate content of both proteoglycans is almost similar, the smaller molecular size and hence the lower overall charge density of the heparan sulfate proteoglycan appears to be partly responsible for its low affinity for LDL. The differences in biologic properties of the two proteoglycans might have implications in the pathophysiology of cardiovascular diseases.

The cultivation of human rotavirus, strain ‘Wa’, to high titer in cell culture and characterization of the viral structural polypeptides

The structural proteins of the ‘Wa’ (serotype 2) strain of human rotavirus have not been described previously. Single-cycle virus growth in MA-104 cells using 5 μg/ml of trypsin in the growth medium was rapid with maximal viral yields (≈10 −6 PFU/ml) obtained 10–12 h post-infection. There was a continuous progression of cytopathic effect (CPE) from 6- to 50-h post-infection. Under conditions of multiple-cycle growth, a greater concentration of trypsin (40 μg/ml) in the growth medium was required to obtain rapid progression of CPE and production of a high titer (≈10 −7 PFU/ml) of infectious (double-shelled) virus. Single- and double-shelled virions were separated by isopycnic centrifugation in CsCl and analyzed by SDS-PAGE. Five proteins with molecular weights of 116,000, 92,000, 88,000, 84,000 and 41,000 were identified as components of the inner shell and four proteins with molecular weights of 60,000, 38,000, 32,000 and 27,000 were located in the outer shell.

Characteristics of the nascent and non-nascent small DNA molecules found in Escherichia coli

We have purified nascent DNA molecules from Escherichia coli pulse-labeled with 5-bromo[6-3H]deoxyuridine by repeated chromatography on nitrocellulose and isopycnic centrifugation in CsCl. The nascent molecules were labeled with 32P either at their 5' ends using polynucleotide kinase or at their 3' ends using terminal transferase. Compared to the non-nascent DNA of normal density, the nascent dense DNA contained a higher proportion of molecules terminated at their 5' ends with ribonucleotides. Exposure of the dense DNA to alkali generated 5' OH termini quantitatively equivalent to the number of molecules bearing 5' ribonucleotides. Experiments designed (1) to detect structures at the 5' ends of phosphatase-treated nascent DNA molecules that caused them to be resistant to hydrolysis by spleen exonuclease or (2) to detect polypeptides that were associated covalently with small DNA molecules and could be iodinated with the Bolton-Hunter reagent did not yield positive results. We conclude that many, if not all, of the intermediates in E. coli DNA replication are initiated with one or more ribonucleotides. The nascent molecules are outnumbered by small non-nascent DNA molecules in the cell, many of which appear to become slightly longer when cells are pulsed with thymidine. Many of the non-nascent DNA molecules behave as if they were self-complementary or crosslinked.

Isolation of arterial wall proteoglycans

Proteoglycans of the arterial media-intima were extracted with associative and dissociative salt solutions. A two step isolation procedure, involving initial precipitation with cetylpyridinium chloride and subsequent CsCl isopycnic density gradient centrifugation was adopted for their effective isolation and purification. The majority of the proteoglycans were: 1. 1. Obtained between buoyant densities 1.60 and 1.77 g ml; 2. 2. Possessed a uronic acid to protein ratio of 2.9 or 1.8 respectively for associatively and dissociatively extracted proteoglycans and 3. 3. Showed a glycosaminoglycan distribution pattern similar to that for the total tissue.

Extraction of cell-associated varicella-zoster virus DNA with Triton X-100-NaCl

Varicella-zoster virus (VZV) DNA was extracted from infected cells with 0.25% Triton X-100-0.2 M NaCl and purified by isopycnic centrifugation in CsCl. In each of eight experiments, 1.8–9.8 μg VZV DNA was obtained from 10 7 infected cells. The VZV DNA obtained by this procedure had a molecular weight of 88–100 × 10 6 as determined by sucrose gradient sedimentation and electron microscopy, and cleavage patterns after digestion with four restriction enzymes that corresponded to patterns previously described with six strains of VZV; the pattern of BamHI-cleaved Triton-NaCl-extracted VZV DNA was identical to the pattern seen after DNA extraction from virions. These studies expand the usefulness of Triton X-100-NaCl for extraction of large molecular weight viral DNA from a system where considerable cell-free virus is produced (Pignatti et al., 1979, Virology 93, 260) to a system known for its marked cell association.

Purification and stability of epizootic hemorrhagic disease virus

Purification of epizootic hemorrhagic disease virus (EHDV) from sonicated cell culture supernatant was effected using polyethylene glycol (PEG) precipitation and two CsCl density gradient centrifugations. During purification procedures the pH was maintained at values close to pH 8.0 as pH stability for EHDV was maximal between pH 7.2 and pH 9.O. During CsCl isopycnic centrifugation at pH 8.0, a 0.1 M or greater buffer was necessary to preserve maximum viral infectivity. This method of purification resulted in the recovery of 75% or more of the infectious virus contained in infected cell culture supernatants. For the extraction of infected cell-associated virus, sonication effectively liberated infectious virus, while freeze-thawing resulted in a significant loss of viral infectivity. The use of Freon allowed for complete recovery of infectious virus while ether or chloroform resulted in lower recoveries of infectious virus.

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [ 14C]- or [ 3H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10 4 and 2.1·10 4 were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with N-[ 3 H]ethylmaleimide or N-[ 14 C]ethylmaleimide , and the 3H to 14C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.

Maize stripe virus: Characteristics of a member of a new virus class

An unusual filamentous nucleoprotein about 3 nm in diameter was consistently associated with maize stripe-diseased maize. Antiserum to purified nucleoprotein neutralized the infectivity of extracts from maize stripe-diseased plants suggesting that the nucleoprotein was the maize stripe virus (MStpV). The rate-zonal sedimentation pattern of the nucleoprotein on sucrose gradients was polydisperse between 51 and 70 S. CsCl isopycnic centrifugation of combined nucleoprotein zones from sucrose gradients resulted in a single band of 1.28 g/ml. The nucleoprotein consisted of 5.2% RNA and a single capsid protein of molecular weight 32,700 daltons. Large quantities of a noncapsid protein of molecular weight 16,300 daltons were also found in MStpV-infected tissue. MStpV was transovarially transmitted by its vector, Peregrinus maidis. Other species susceptible to MStpV in addition to Zea mays were Rottboellia exaltata and Sorghum bicolor. The similarities between MStpV and rice stripe virus are discussed. We conclude that these viruses represent a new virus class.

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The relative efficiencies of various methods of concentration and purification of the eel virus of Europe (EVE) were examined.Whilst Freon treatment had no obvious effect on yields, EVE was effectively concentrated 10 times using polyethylene glycol (PEG), and could be concentrated 100 times using a 2 step PEG technique, or by use of high speed centrifugation as alternative to the 2nd PEG step. High speed centrifugation had the advantage of excluding UV absorptive substances other than virus, which could not be excluded by the 2 step PEG method.EVE was partially purified by sucrose linear 15-45% density gradient centrifugation at 80, 000g for 2 hrs. Well purified EVE was obtained by CsCl isopycnic centrifugation at 160, 000g for 24 hrs at a bouyant density of 1.33g/cm3. IPNV-VR299 was concentrated and purified in a similar manner to EVE and was also shown to have a density of 1.33g/cm3. The mean diameter of uranyl-acetate stained EVE was 65.5nm and that of IPNV 66.6nm. CsCl isopycnic centrifugation of EVE, but not of IPNV, revealed a faint band at a density of 1.30g/cm3 composed of many particles of average diameter 20nm apparently morphological subunits.This feature, together with the preserve of empty and collapsed particles in the main EVE band, suggested a major difference between EVE and IPNV in virus stability.