Some factors affecting infectivity assays of wound-tumor virus on cell monolayers from an insect vector

Measurements of wound-tumor virus (WTV)-infectivity were made by counting infection foci in cell monolayers of Agallia constricta. The optimum seeding density was about 2.9 × 10⁵ cells per 0.1 ml per coverslip. The foci were revealed by staining with fluorescent antibody and examination with the fluorescence microscope. The focus count method employed in these studies, is shown to be an accurate method for determining WTV infectivities. The number of infected cells per focus was related to the concentration of WTV in the inoculum and is probably an expression of the rate of virus multiplication and spread in the cell monolayer. The effects of glycine, histidine, phosphate, and MgCl₂ in inocula were studied; a histidine-MgCl₂ (His-MgCl₂) solution was best for experiments with WTV on cell monolayers. Tumor extracts diluted to 10⁻² in His-MgCl₂ solution were quick-frozen with no detectable loss in infectivity and maintained a constant infectivity at −80° for at least 9 months. The optimal temperature for thawing was about 37°. Optimal inoculation and incubation periods at 30° were dependent upon the WTV-concentration in His-MgCl₂ solution. Optimal periods of inoculation with 8.3 × (10^7.5, 10^7.0, and 10^6.5) virions per milliliter were 1.5, 2, and 3 hr, respectively; optimal incubation periods for the same concentrations were 36, 42, and 54 hr, respectively. A satisfactory assay procedure for inocula containing about 8.3 × 10^7.0 to 8.3 × 10⁶ virions per milliliter consisted of applying 0.01 ml of inoculum per coverslip for 1 hr at 30°; after this an incubation period of 40–60 hr was allowed before focus counts were made.     

Improved method for the purification of Hog Cholera Virus grown in tissue culture

Summary A method for purification of Hog Cholera Virus (HCV) is presented. Cell-associated virus was extracted from PK₁₅ cells 17 hours p.i. by fluorocarbon treatment. The virus was concentrated by polyethylene glycol precipitation and partially purified by pelleting on a fluorocarbon cushion. Final purification was achieved by rate zonal centrifugation in a 7–35 per cent sucrose gradient. This procedure permits a recovery of approximately 40 per cent of viral infectivity. A specific infectivity of about 2×10⁷ PFU/µg of protein was achieved. An apparent density d=1.13 g/ml in sucrose and a S_w ²⁰ value of about 150 was determined for purified HC virions.With 6 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.     

Purification and separation of enveloped and unenveloped herpes simplex virus particles.

Enveloped and unenveloped forms of herpes simplex virus (HSV) occurring in infected rabbit lung (ZP line) cells were purified by differential and discontinuous Ficoll density gradient centrifugation. Then the viral particles were separated in a sucrose-D2O density gradient. In the course of the procedures, both virus preparations were freed of Mg2+-dependent Na+ plus K+-stimulated adenosine triphosphatase (ATPase), 5'-nucleotidase, and glucose-6-phosphatase activities. However, Mg2+ -activated ATPase was shown to be firmly associated with purified virions. The recovery of infectious virus was 50-60 percent. The specific infectivities (TCID50/mg protein) of the purified enveloped and unenveloped viral particles were 1-2 times 10(10) and 2-5 times 10(6), respectively. The infectivity of the unenveloped viral particles was discussed.     

Some properties of clover blotch virus.

Clover blotch virus (CBV) was purified by differential centrifugation or precipitation with polyethylene glycol 6000 or 20000. Analytical centrifugation of purified CBV preparations revealed 2 components, the proportions of the lighter (96 S) nad heavier (112 S) being 88 and 12%, respectively. RNA isolated from purified CBV represented a micture of several RNA species of different molecular weight and its infectivity was by about 2 log units lower than that of the original nucleoprotein. CBV virions are isometric with a diameter of 28 nm. CBV is tentatively included into the Cucumovirus group.     

Degradation of brome mosaic and tobacco mosaic viruses in bentonite

Brome mosaic virus degraded to RNA in 0.02 M tris buffer, 0.001 M EDTA, pH 7–9, containing bentonite and 0.1 M NaCl. The virus coat protein was apparently adsorbed by the bentonite. In bentonite in buffer without NaCl, brome mosaic virus degraded to three nucleoproteins, each containing one piece of RNA. The largest nucleoprotein was infective. Tobacco mosaic virus degraded to RNA and protein in the above buffer with bentonite, but degraded only to short, variable length rods when 0.1 M NaCl was added. Tobacco mosaic virus coat protein apparently was not adsorbed by the bentonite. Tobacco mosaic virus RNA prepared by bentonite treatment and density-gradient centrifugation had about 20% the specific infectivity of RNA in intact virions.     

Double-stranded nucleic acids found in tissue infected with the satellite of tobacco ringspot virus

We have purified a multicomponent population of RNAs from tissue infected with the satellite of tobacco ringspot virus (S-TRSV) that is not present in tobacco ringspot virus-(TRSV-)infected tissue or in uninfected tissue. Many of the properties are characteristic of dsRNA, or the so-called replicative form of small RNA viruses; i.e., a sharp melting profile at relatively high temperature and high hyperchromicity and buoyant density in cesium sulfate; the RNAs are infective only after denaturation and quick quenching, followed by addition of TRSV. The infectivity is not destroyed prior to denaturation either by pancreatic RNase (in high ionic strength buffer) or by incubation with formaldehyde. Other properties are not typical of dsRNAs: At least 83% of the RNAs elute from CF-11 cellulose columns in buffer (0.1 M NaCl, 0.05 M Tris, 0.001 M EDTA, pH 6.9)/ethanol mixtures that typically elute ssRNAs but not dsRNAs. The RNA is composed of many components, some of which are up to 20 times the mass one would expect from the known mass of the corresponding RNAs found in S-TRSV virions. Pancreatic RNase, at relatively high concentrations, converts these higher-molecular-weight dsRNAs into dsRNAs of lower molecular weight. These lower-molecular-weight double-stranded components retain a high level of infectivity after denaturation (with added TRSV).     

Purification of pea enation mosaic virus from its pea aphid vector, Acyrthosiphon pisum, and aphid-transmission characteristics

Pea enation mosaic virus (PEMV) was purified from pea aphids by a system involving chloroform-butanol emulsification, differential centrifugation, and density gradient centrifugation. The product was highly infectious when transmitted to pea plants by pea aphids, and it was extracted in sufficiently high concentration to be monitored in centrifuged sucrose columns with a UV scanner. Electron micrographs showed that purified preparations contained an abundance of virus particles that measured ca. 27 nm in diameter and were indistinguishable from those isolated from infected plants. No virus particles were detected in preparations of nonviruliferous aphids, and no infectivity was obtained from these preparations in either aphid or mechanical transmission tests.Virus purified from an aphid source was established in pea plants by mechanical transmission and compared as to aphid-transmissibility with a virus line from a plant source; both lines were initiated with the same PEMV strain. The aphid-source virus line was transmitted by pea aphid first-stage nymphs with significantly (P < 0.05) higher efficiency than the plant-source virus line after 1- and 4-hr acquisition-access periods, and it was transmitted with characteristics superior to those previously recorded for PEMV-pea aphid relationships. First-instar pea aphids were 95.8% efficient in transmission of the aphid-source virus after only a 4-hr acquisition-access period on infected pea plants. The virus line when acquired from infected plants had a median latent period (LP₅₀) in the first instar of only 5.7 hr.     

Purification and antigenicity of three isolates of barley yellow dwarf virus

An isolate (RPV) of barley yellow dwarf virus transmitted specifically by Rhopalosiphum padi and an isolate (PAV) transmitted nonspecifically by both R. padi and Macrosiphum avenae were purified by procedures previously found satisfactory for another isolate (MAV) transmitted specifically by M. avenae. As with MAV, infectivity of RPV and PAV samples removed from sucrose gradient columns was associated with a dense polyhedral particle about 30 nm in diameter in shadowed preparations. No differences in sedimentation rate (sedimentation coefficient 115–118 S) among the 3 virus isolates were detected in parallel sucrose gradient centrifugation tests.     

The comparative susceptibilities of cultured vector and nonvector leafhopper cells to three plant viruses

The susceptibilities of established leafhopper cell lines from Agallia constricta (AC), Aceratagallia sanguinolenta (AS), and Agalliopsis novella (AN) were compared with one another when inoculated with wound tumor virus (WTV), the constricta variety of potato yellow dwarf virus (CYDV), and the sanguinolenta variety of potato yellow dwarf virus (SYDV). The comparative susceptibility of an established cell line from Dalbulus elimatus (DE) to these viruses was also determined. D. elimatus is not closely related to agallian leafhoppers nor has it been shown to be a vector of any of the above viruses. Vector AC cells and AN cells were about equally susceptible to WTV. The susceptibility of nonvector AS cells to WTV was about 0 · 064 times that of the vector AC and AN cells. Despite the insect-virus relationships, all cell lines derived from agallian leafhoppers were about equally susceptible to CYDV and SYDV. Concentrations of WTV and CYDV which gave about 25% infection on vector AC cells failed to infect DE cells. On the contrary, SYDV infected the nonvector DE cells although the incidence of infection was only about 0.0027 times that of vector AS cells.     

Purification of rabies virus from tissue culture

Summary A procedure for the purification of rabies virus grown in BHK-21 C 13 cells was developed that consisted of batch adsorption of the virus to aluminium phosphate gel equilibrated with 0.05m phosphate buffer at pH 7.1–7.4 and elution from the gel with 0.3m phosphate buffer at pH 8.0. On average 75 per cent of viral infectivity and 25 per cent of hemagglutinating activity were recovered. Purification factors of about 2000 were achieved as indicated the comparison of the protein contents of original and purified virus samples with their biological activities. On average purified preparations contained 10¹¹ LD₅₀, 2 × 10⁵ hemagglutinating units and 3×10⁴ complement fixing units per mg of protein. Immunization of rabbits with AlPO₄-purified virus produced potent rabies antisera which reacted with extracts of BHK-21 cells to a neglegible extent but not with bovine serum albumin. In CsCl gradients virus infectivity banded at densities of 1.22 and 1.20 g/ml.     

Some properties of TYMV nucleoproteins isolated in cesium chloride density gradients

The proportion of the noninfectious nucleoprotein with a normal amount of RNA (B₂ fraction) found in turnip yellow mosaic virus (TYMV) preparations following fractionation in CsCl density gradients, increased from about 15% to 50% as the pH of the CsCl solution was raised from 7.0 to 8.5. The other minor nucleoprotein fractions were not increased in this way.When the pH of the CsCl was controlled in the range 5.0–7.0, and the SW 40 rotor was used, double bands of each of the previously described nucleoprotein components were revealed. Thus ten nucleoprotein bands could be observed, two each for B₀₀₀, B₀₀, B₀, B₁, and B₂. The less dense band (_a band) of each pair was present in greater amounts. Both the bands found in the component previously shown to contain infectivity (B₁) were infectious. Mixing these isolated bands (B_1a and B_1b) did not enhance infectivity.Double bands were formed by some preparations of empty protein shells when these were subjected to centrifugation in CsCl of D = 1.27 g/cm³. No consistent differences were found in the behaviour of the polypeptide from a and b bands on polyacrylamide gel electrophoresis.     

Separation of herpes simplex virus virions and nucleocapsids on Percoll gradients

Herpes simplex virus (HSV) virions and nucleocapsids were separated and purified by centrifugation in density gradient of polyvinylpyrrolidone-coated colloidal silica (Percoll). Virions and nucleocapsids banded at densities of 1.07 and 1.03 g/ml, respectively. The distribution in the gradient of virions and nucleocapsids suggested that particles were discriminated according to difference in size rather than in density. The reduction of cell proteins in preparations of purified virions was 1300--2100 times. The recovery of infective virus was approximately 30%.     

Purification and biophysical properties of human coronavirus 229E

Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 10^9.0–10^9.5 TCID₅₀/ml and plaque titers from 10^10.2–10^10.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO₄ gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E_1cm^1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S_20,v⁰ = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of ³H- and ¹⁴C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.     

Serological studies on virions and polyhedron protein of a nuclear polyhedrosis virus of the cabbage looper, Trichoplusia ni.

Improved purification procedures were employed to isolate polyhedron protein and enveloped virus particles (virions) from a nuclear polyhedrosis virus (NPV) of the cabbage looper, Trichoplusia ni, to study the serological relationship between these two components. Polyhedra were digested in sodium carbonate-saline buffer, pH 11, for 1 hr at room temperature and centrifuged at 40,000 g for 30 min to crudely separate the polyhedron protein from the virions. Polyhedron protein was purified by Sepharose 6B column chromatography and virions were purified by Ficoll density gradient centrifugation and Sepharose 2B column chromatography. Antiserum was prepared by injecting rabbits with highly purified intact polyhedra, polyhedron protein, or virions. When partially purified antigens were used in Ouchterlony double diffusion in agarose gels, multiple precipitin lines were formed and polyhedron protein cross-reacted with virions. However, the highly purified polyhedron protein and virions reacted only with their homologous antisera and no cross-reactions were visible. Immunoelectrophoresis showed that virions and polyhedron protein had significantly different mobilities and reacted only with their homologous antisera. It was therefore concluded that no serological relationship exists between highly purified polyhedron protein and virions of NPV of T. ni.     

Effects of sialylation of influenza virions on their interactions with host cells and erythrocytes

Influenza virions can be sialylated by incubating purified virus with sialyltransferase and CMP-sialic acid. Sialic acid residues are added only to the virion hemagglutinin. Both portions of the hemagglutinin subunit have terminal galactose residues which accept sialic acid, with the HA₁ region being preferentially sialylated early in the reaction. The rate of sialylation depends on the source of the virus; virions derived from chick embryo fibroblasts are sialylated at approximately 20 times the rate of virus from an established line of bovine kidney cells. Sialylation destroys the hemagglutinating activity of the particles but leaves the neuraminidase activity unchanged. The ability of these preparations to form plaques on MDBK cells is either enhanced or unchanged depending on the amount of sialic acid added to the virions. Maximum enhancement is observed when approximately 1250 sialic acid residues are added per virion. Addition of more residues decreased the amount of enhancement. Heavily sialylated virus shows the same infectivity as unsialylated virus although it has an isoelectric point below pH 4.0; that of unsialylated virus is 6.0 to 6.5. In an attempt to determine the basis for the loss of hemagglutinating activity and the enhancement of infectivity, we have measured the ability of sialylated and control virions to bind to erythrocytes and to host cells under conditions of either virus or cell excess. Identical results are obtained with sialylated and control virus at all virus to cell ratios tested, indicating that the changes induced by sialylation do not reflect changes in the ability of virus to bind to cellular receptors. The results suggest that the formation of infectious units by aggregation of randomly defective particles along with changes in step(s) following binding are responsible for the observed alterations in biological activities.     

The stabilization and purification of respiratory syncytial virus using MgSO4

Respiratory syncytial (RS) virus infectivity is stabilized by MgSO₄. In the presence of 1 M MgSO₄ at 4°, only 50% of viral infectivity is lost in 12 weeks. This is a 30-fold increase in RS virus stability. The increased stability of RS virus allowed infectivity to be used as a convenient marker for the presence of RS virus during multiple cycles of density gradient centrifugation.     

Specific infectivity of different wound tumor virus isolates

The specific infectivities of certain wound tumor virus (WTV) strains for vector cell monolayers were determined on the basis of counts of virions in droplet residues in the electron microscope and on the numbers of infections they produced on vector cell monolayers. Infectivity for vector cell monolayers is lost completely in 5 to 9 years if an isolate of WTV is maintained in sweet clover without transmission by the vector. This loss does not occur as a single, all-or-nothing change but involves several step-like losses. It is accompanied by a decline in virus concentration in the tumois, but the depreciation of specific infectivity is the principal cause of the loss of infection for vector cell monolayers. The probability that these losses also cause corresponding losses of ability to be transmitted by the vector is discussed.     

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.