Comparison of influenza and parainfluenza RNP properties

Summary Fowl plague virus and Sendai virus were disrupted by Tween 20 in an alcaline medium. Whereas in the case of fowl plague virus sedimentation of the ribonucleoprotein (BNP) in sucrose gradients revealed three components sedimenting at 64S, 58S, and 48S, the RNP of Sendai virus showed a sedimentation coefficient of 110S only. During buoyant density studies in CsCl the three components of fowl plague virus RNP banded at the uniform density of 1.35–1.36 g/cm³ and the RNP of Sendai virus was found at a medium density of 1.32 g/cm³.     

Separation of ECHO virus type 6 antigens by centrifugation in caesium chloride density gradients

Summary ECHO virus 6 particles separated by caesium chloride density gradient centrifugation were studied by electron microscopy. Particles in fractions of a density of 1.33 g/cm³, carrying peak infectivity and N antigenicity, appeared in the electron microscope as complete virions. The diameter was found to be 25 m. Particles in fractions of a density of 1.30 g/cm³, carrying H antigenicity, appeared as more or less empty capsids. This was true for the naturally occurring H antigen as well as for H antigen obtained by heat treatment.     

The RNA of the human syncytium-forming (foamy) virus

Summary Human syncytium-forming (foamy) virus was labeled with³H-uridine and banded isopycnically in sucrose gradients (buoyant density = 1.16 to 1.18 g/cm³). Viral RNA extracted from the banded virus was analyzed either by rate zonal separation in sucrose gradients or by polyacrylamide-agarose gel electrophoresis. The results indicated that purified HSFV contains a 60S RNA component plus several smaller molecular weight RNA components. On dissociation with heat, smaller RNA structures were released from the 60S component. These results indicate that the genome of HSFV, like the other members of theRetroviridae family, is composed of an aggregate of several RNA species.With 3 Figures     

Purification and some physicochemical properties of sonchus yellow net virus

Sonchus yellow net virus (SYNV) was purified from a Nicotiana hybrid by Celite filtration and sucrose density gradient centrifugation. Infectious preparations sedimented at 1044 S in linear-log gradients and banded at 1.183 g/ml in sucrose equilibrium gradients. Electron microscopy of purified preparations revealed bacilliform particles (94 × 248 nm). The virions had internal cross striations with a periodicity of about 4.1 nm and surface projections about 6 nm long. The molecular weight of the virion, estimated from size and density, was about 9 × 10⁸. Nucleic acid from sodium dodecyl sulfate-disrupted virions was susceptible to RNase, sedimented in sucrose gradients at 44 S, and had a molecular weight of 4.42 × 10⁶ as estimated by polyacrylamide-gel electrophoresis. Four major polypeptides with average molecular weights of 76,800, 63,800, 45,500, and 39,500 were detected by gel electrophoresis. SYNV preparations reacted in gel diffusion tests with a homologous antiserum but not with antisera to broccoli necrotic yellows virus, lettuce nectrotic yellows virus, or sow thistle yellow vein virus.     

Characterisation of rubella virus hemagglutinin rosettes

Purified rubella virus treated with Triton X-100 was subjected to centrifugation in a sucrose density gradient containing nonionic detergent β-D-octylglucoside. The result of this treatment was the formation of hemagglutinating rosettes containing viral glycoproteins VP₂ (50,000 mol. wt.) and VP₃ (63,000 mol. wt.). The rosettes have a 26 S sedimentation coefficient and a density of 1.2 g/cm³ in sucrose. Electron microscopy revealed 15 nm rosettes with a hollow center. The molecular weight of the rosettes was extrapolated at 850,00     

Abortive infection of influenza virus in Ehrlich ascites tumor cells. Unusual fragility of virus particles

Summary Noninfectious virus particles were produced in Ehrlich ascites tumor cells infected intraperitoneally with fowl plague virus. The PFU yield of virus per cell was less than 0.1 and the ratio PFU/HA units in the progeny virus was less than 10³. The virus particles had the same morphology and size as egg-grown virus but were more fragile. They were disrupted by centrifugation through sucrose and caesium chloride gradients, but this disruption was avoided by fixing the particles with formaldehyde before centrifugation. Analysis of polypeptides by SDS-PAGE showed that ascites-grown virus particles contained reduced amounts of matrix protein compared with egg-grown virus.With 6 Figures     

Properties of mouse leukemia viruses. VII. The major viral glycoprotein of friend leukemia virus. Isolation and physicochemical properties

The major viral glycoprotein of Friend leukemia virus was isolated by a procedure comprising density gradient centrifugation, Con A affinity chromatography, and Sephadex gel filtration. It was obtained in milligram amounts and found to be pure by physicochemical and seroimmunological methods. It has a buoyant density in sucrose of 1.18 g/cm³ and a molecular weight of 71,000 as determined by SDS-polyacrylamide gel electrophoresis. A molecular weight of 58,000 was calculated from s₂₀ = 4.05 S and D₂₀ = 6.5 · 10^?7 cm²/sec. The specific complement-fixing activity of the purified material was 50,000–100,000 complement-fixing units/mg protein.     

Purification and characterization of bovine herpesvirus-1 isolates and virus DNA utilizing bovine embryonic lung cells

Summary Techniques are described for the culture of bovine embryonic lung (BEL) cells by the primary explant technique and mild enzymatic disaggregation of lung tissue. Preparations of bovine herpesvirus-1 (BHV-1) isolates with titers of 10⁹ plaque-forming units/ml are purified by sucrose and cesium chloride gradient centrifugation after replication in primary cultures of BEL cells. BHV-1 isolated by velocity sedimentation bands in 35% sucrose and sediments at a density of 1.23 g/cc in cesium chloride density gradients. The DNAs of all virus isolates have a velocity sedimentation coefficient of approximately 54S. Virus DNAs from all isolates sediment to equilibrium in cesium chloride at 1.730 g/cc. Differences between virus isolates can be demonstrated by digestion of virus DNA with the restriction endonucleaseKpnI and by polyacrylamide gel electrophoresis of virion polypeptides. These techniques are useful for production of purified high titer virus free of host cell contaminants.     

Buoyant density in cesium chloride of the human reoviruslike agent of infantile gastroenteritis by ultracentrifugation,electron microscopy,and complement fixation

The buoyant density in cesium chloride of the human reoviruslike (HRVL) agent of infantile gastroenteritis was studied utilizing electron microscopy and complement fixation (CF) for the detection of reoviruslike particles in fractions of the density gradient. Three virus positive stool filtrates were studied. “Full” reoviruslike particles had a density of 1.35–1.37 g/cm³, whereas “empty” particles had a density of 1.29 g/cm³. Peak CF activity coincided with the peak fraction of both the “full” and “empty” reoviruslike particles. In addition, by differential centrifugation, CF activity was also associated with the virion and not a “soluble” antigen.     

3' terminal nucleotide sequence of human astrovirus type 1 and routine detection of astrovirus nucleic acid and antigens.

Human astrovirus type 1 was purified by caesium chloride density-gradient centrifugation and the virus was located using an immunodot blot technique with polyclonal rabbit serum, which reacted with all five serotypes. The virus banded with a density of 1.33 g/ml. RNA was extracted from the purified virus, converted into double-stranded cDNA, using an oligo(dT) primer, and cloned into plasmid and M13 vectors. The sequence of the 3' end of astrovirus RNA adjacent to the poly(A) tract was determined. This sequence showed no significant homology with the equivalent region of other positive-sense RNA viruses. Synthetic oligonucleotide primers were designed to amplify specifically astrovirus type 1 RNA in a polymerase chain reaction.     

Biochemical studies with infectious bursal disease virus: Comparison of some of its properties with infectious pancreatic necrosis virus

Summary Infectious bursal disease (IBD) virus was purified from the bursae of infected chickens. Two morphologically indistinguishable populations of virus particles were separated in sucrose gradients and possessed sedimentation coefficients of 395S and 460S. Both populations contained RNA and had identical polypeptide compositions. IBD virus banded at a density of 1.31 g/ml in CsCl and at 1.24 g/ml in sodium potassium tartrate.IBD virus contained two RNA segments with mol. wts. of 2.4×10⁶ and 2.2×10⁶ as estimated by polyacrylamide-agarose gel electrophoresis, but sedimented in sucrose gradients at 15S. Virus RNA was resistant to 0.1 µg/ml ribonuclease treatment under conditions in which ribosomal RNA was completely hydrolysed, but was sensitive to 1.0 and 10 µg/ml treatments. These results suggest that the RNA consists of either double-stranded or highly ordered single-stranded molecules. IBD virus contained seven polypeptides with mol. wts. in the range 97,000 to 24,000. Two polypeptides were absent in empty particles of IBD virus.IBD and infectious pancreatic necrosis (IPN) viruses were morphologically indistinguishable. IPN virus possessed a sedimentation coefficient of 440S and banded at a density of 1.32 g/ml in CsCl. In addition the electrophoretic mobilities of IBD and IPN virus RNAs were almost identical. Polyacrylamide slab gel electrophoresis showed that while the number and size of the polypeptides were different for each virus there were similarities in the overall pattern.With 5 Figures     

Evidence for endogenous reovirus-like particles in a tissue culture cell line from Drosophila melanogaster

Virus-like particles were isolated from the Blood IIIBlood III cell line of Drosophila melanogaster by sucrose and cesium chloride density centrifugation. The particles have a buoyant density of 1.37 g/cm³ in cesium chloride and a diameter of 568 Å The viral genome contains double-stranded RNA by the criteria of resistance to ribonuclease A and nuclease S₁. Polyacrylamide gel electrophoresis resolves the RNA into 10-segments and an additional minor component rangina in molecular weight from 0.55 × 10⁶ to 2.75 × 10⁶.     

Physicochemical properties of two distinct types of virus-like particles from Helminthosporium victoriae

Two serologically distinct types of virus-like particles (VLPs) were isolated from Helminthosporium victoriae. The first, which was isolated from three normal and two diseased isolates, sedimented at a rate of 190 S when centrifuged in linear-log sucrose density gradients. The second, which was found only in the two diseased isolates, had a sedimentation coefficient of 145 S. No VLPs were detected in two other normal isolates of the fungus. The 190 and 145 S VLPs both were polyhedral and 35 to 40 nm in diameter and had typical nucleoprotein absorbancy spectra. The 190 S type of VLP was electrophoretically distinct from the 145 S VLP when electrophoresed on either polyacrylamide gels or cellulose acetate strips. Equilibrium density gradient centrifugation in cesium chloride revealed a single density component for the 190 S VLPs with a buoyant density of 1.4325 g/cm³ The 145 S VLPs could be resolved into two to four components with buoyant densities of 1.3813 to 1.4138 g/cm³. SDS polyacrylamide gel electrophoresis of the 190 S VLPs revealed two major proteins of molecular weight 83,000 and 88,000 present in equimolar amounts. The 145 S type of VLP had three major proteins with molecular weights of 92,000, 97,000, and 106,000; these were also present in equimolar amounts. Both the 145 and 190 S VLPs contained double-stranded RNA (dsRNA). The 190 S type of VLP had a single species of dsRNA with a molecular weight of 3.0 × 10⁶. There were four classes of dsRNA associated with the 145 S VLPs, and their molecular weights were 2.4, 2.2, 2.1, and 2.0 × 10⁶.     

Structural polypeptides of the enteropathogenic bovine coronavirus strain LY-138

Summary The bovine coronavirus strain LY-138 was purified by differential as well as velocity and isopycnic centrifugation in sucrose or CsCl gradients. The substrate for purification was contents of the small intestine of experimentally inoculated calves. This strain is highly enteropathogenic, but it could not yet be propagated in cultured cells. Intact virions had a density of 1.245 g/cm³ in CsCl and 1.185 g/cm³ in sucrose. A spherical core-like structure with an average diameter of 82 nm remaining after treatment with chloroform had a density of 1.299 g/cm³ in CsCl and 1.201 g/cm³ in sucrose.Seven distinct bands of polypeptides and 4 shoulders were detected after electrophoresis of SDS-solubilized virions in polyacrylamide gels. The approximate molecular weights ranged from 110,000 to 36,000. Four of the bands gave a PAS positive reaction. These 4 glycoproteins and an additional protein with an approximate molecular weight of 70,000 were removed by chloroform treatment. The remaining core-like structure contained the 2 polypeptides VP3 and VP7.With 5 Figures     

Subfractionation of CsCl-purified H-1 parvovirus on metrizamide gradients.

The different density classes of H-1 parvovirus, collected within 30 hr of parection of parasynchronous cultures, following the standard CsCl purification step, have been shown to be heterogeneous. Rebanding of the denser form (HF, ? = 1.46 g/cm³) and the less dense form (LF, ? = 1.42 g/cm³) of infectious virus in the nonionic density generating solute, metrizamide, showed that both HF and LF virus bands were heterogeneous in density. The infectivity banded with isotopically labeled virus protein and DNA at 1.32 g/cm³ for both HF and LF virus. Amounts of protein and DNA which varied from preparation to preparation, but which were greater from the HF virus band, were distributed throughout the rest of the gradient, but predominated in a peak at a density of 1.2 g/cm³. The protein in this peak was without hemagglutinating activity but had the molecular weights and proportions of the H-1 virion proteins (VP1, VP2′, and VP2). The DNA was of the same size as H-1 DNA monomers and its proportion to the protein was similar to that of the infectious peak. The DNA was susceptible to micrococcal nuclease digestion. The nature of this noninfectious viral material thus seemed to be incompletely assembled virus. Radio-labeled H-1 virus collected after 72 hr of infection formed a discrete single peak in both CsCl (? = 1.42 g/cm³), and metrizamide gradients (? = 1.32 g/cm³). There was no significant amount of the 1.20 g/cm³ viral protein-DNA complex in these mature preparations.     

The genome of equine arteritis virus.

Equine arteritis virus (EAV) contains an infectious RNA. [³H]uridine-labeled RNA was released from purified virus (density, 1.155 g/ml in sucrose; s_20,w, 224 ± 8 S) with sodium dodecyl sulfate and 2-mercaptoethanol. An s_20,w value of 48 S was found in isokinetic sucrose gradients in 0.1 M saline. In 1 mM saline, sedimentation was slower (33 S), in 0.1 M saline plus 1 mM MgCl₂, a value of 56 S was measured. A molecular weight of 4.0 × 10⁶ was determined by polyacrylamide-agarose-gel electrophoresis. Heating of purified RNA in the presence of 1.1 M formaldehyde and subsequent centrifugation in gradients containing formaldehyde did not result in degradation to smaller RNA's. From this procedure a molecular weight of 4.1 × 10⁶ was calculated. Buoyant density in Cs₂SO₄ of the RNA was 1.65 g/ml. In most experiments Semliki forest virus RNA was taken as a reference. It behaved almost indistinguishably from the RNA of EAV. In conclusion EAV contains an infectious, colinear molecule of single-stranded RNA with a molecular weight of about 4 million. These data justify a definite inclusion of this virus in the family Togaviridae.     

Purification studies of rubella virus

Summary Equilibrium centrifugation of rubella virus in potassium citrate density gradients revealed a heterogeneous population of virus particles, with 90% of the infectivity being recovered at a density of 1.20g/cm³ and the remaining 10% at 1.10 g/cm³. Pretreatment of the virus material with 1% Tween 80 gave a homogeneous virus peak at 1.20 g/cm³, presumably by splitting aggregates of virus and cell debris.Chromatography through DEAE Sephadex A25 revealed two distinctly different virus fractions, with 10% appearing in the void volume and 90% being eluted with 0.3–0.5M MgSO₄. These fractions were shown not to be influenced by pretreatment with Tween.This work was supported by grant Nr. 65-77 from the Swedish Foundation for Cancer Research.     

Purification of a herpes-like virus from abalone (Haliotis spp.) with ganglioneuritis and detection by transmission electron microscopy

A herpes-like virus was for the first time purified from abalone diagnosed with ganglioneuritis. Pleuropedal ganglia, pedal nerve cords, head and epipodial tissue was collected and homogenized from abalone populations exhibiting high mortality and clinical signs consistent with herpes-virus like ganglioneuritis. Following ultracentrifugation by sucrose gradient prepared in sea-water, the purified virus was negatively stained and examined under a transmission electron microscope. Virus particles were observed to have an icosahedral capsid appearance surrounded by an envelope with numerous spikes on the external surface. The capsid ranged 92-109 nm in diameter and the enveloped virus was approximately 150 nm in diameter. Virus particles were found mainly at the interface of 40-50% sucrose gradients, and a few presented at the interface of 50-60% sucrose gradients. Isopycnic gradient centrifugation was performed in a potassium tartrate gradient and caesium chloride gradient, where the buoyant density of the herpes-like virus was determined to be 1.17-1.18 g/mL. The use of sea-water as the buffer in preparation of the gradient was critical in the preliminary purification of the herpes-like virus, and more efficient harvesting of the virus was achieved by sucrose and potassium tartrate gradients than caesium chloride gradient. The described method, whilst proving successful for purifying a herpes-like virus from abalone, may also be applicable to other viruses from marine animals.     

In vitro growth characteristics and heterogeneity of mouse hepatitis virus type 3

Summary Thein vitro virus yield of MHV3 reached 10⁷ PFU/ml in mouse DBT cells infected with a virus suspension in HEPES-buffered medium containing DEAE-dextran. The virus titer was 10⁶ PFU/ml in the presence of 10 µg actinomycin D/ml. MHV3 grown in DBT cells gave three peaks of density (1.10–1.14 g/cm³, 1.18–1.20 g/cm³, and 1.25–1.31 g/cm³) in sucrose gradients. All these peaks retained infectivity.With 2 Figures     

Chick embryo lethal orphan (CELO) virus: some physical and immunological properties

Chick embryo lethal orphan (CELO) virus was partially purified by equilibrium centrifugation in CsCl density gradients. The virus was found to band at 1.32 g/cm³, and this band was found to contain the peak infectivity titres of virus. Electron microscopy of partially purified CELO virus revealed icosahedral particles of 60–80 nm diameter, and with a capsid of 252 capsomeres. The thermal denaturation temperature (T_m) of CELO virus DNA indicated a base composition of 46% guanine-cytosine. CELO virus CF antigens, separated from virus particles during purification of the virus, had the same sedimentation properties in preformed linear sucrose gradients as did the CF antigen of adenovirus 7.