Transformation Potentials of the Noninfectious (Defective) Component in Pools of Adenoviruses Type 12 and Simian Adenovirus 7

Pools of adenovirus 12 and simian adenovirus 7 were separated into four or five fractions by density gradient centrifugation in cesium chloride. Each fraction was analyzed for total in vitro infectivity units, total transformation activity, and for total virus particle (VP) content. Two major subpopulations were separated with mean densities of 1.30 +/- 0.02 and 1.34 +/- 0.02 g/ml, respectively. Virions in the 1.34 g/ml range were highly infectious (10(2) to 10(3) VP per infectivity unit) in contrast to virions at 1.30 g/ml density (10(4) to 10(5) VP per infectivity units). Transformation capacity was evenly distributed throughout fractions of both viruses, indicating that genetically incomplete or defective virus particles were not deficient in their ability to induce transformation. The average VP per transformation unit for adenovirus 12 (2.85 x 10(6)) and for simian adenovirus 7 (4.00 x 10(6)) did not vary significantly from fraction to fraction. These values were obtained with optimal input multiplicities of 16 to 64 VP per cell. At higher multiplicities the apparent increase in VP per transformation unit was attributable to the viral cytocidal effect on hamster cells. These studies revealed that quantitation of in vitro transformation based on VP multiplicities was more reliable than on the basis of infectious units. These estimates were independent of method of virus production, extraction, and purification.     

Characterization of the Kilham Rat Virus

Kilham rat virus (KRV) was found to grow in a rat nephroma cell line and to form plaques on secondary rat embryo monolayers. The virus was purified by enzymatic treatment and isopycnic cesium chloride sedimentation. KRV bands at a density of 1.41 g/cm(3) in cesium chloride. It contains about 26.5% deoxyribonucleic acid (DNA). The sedimentation coefficient S(20,w) in sucrose gradients was 122 corresponding to a molecular weight of 6.6 x 10(6) daltons. The reaction of formaldehyde with the KRV virion suggests that the DNA in situ is single-stranded. DNA extracted from KRV had a buoyant density of 1.715 g/cm(3) in cesium chloride. The S(20,w) was determined in sucrose gradients to be 16, and the molecular weight was calculated to be approximately 1.7 x 10(6) daltons. The base composition of the DNA is 26.7% adenine, 30.8% thymine, 20.0% guanine, and 22.5% cytosine. On the basis of its noncomplementary nucleotide ratio, melting curve, and the reaction with formaldehyde, the DNA of KRV is believed to be single-stranded.     

Evidence for secondary structure within the virion RNA of echovirus 22.

Phenol-extracted echovirus 22 virion RNA is infectious, but unlike poliovirus virion RNA, it resists digestion with pancreatic RNase and nuclease P-1, a 3' exonuclease selective for single-stranded RNA. These data indicate the presence of an enzyme-resistant portion somewhere in the RNA molecule and suggest that it is a double-stranded or base-paired region distant from the unblocked 3' terminus. Equilibrium density gradient centrifugation of native echovirus 22 virion RNA results in a single peak with a density of 1.63 g/cm3. When sheared before centrifugation, the molecule is resolved into two RNA species: one with an approximate density of 1.70 to 1.71 g/cm3, as is observed also for single-stranded poliovirus virion RNA, and the other with a density of 1.58 to 1.59 g/cm3. Data obtained from rate zonal centrifugation may be used to calculate an approximate sedimentation coefficient corrected to water at 20 degrees C of 34 and a molecular weight of 2.4 X 10(6) for the virion RNA. We propose a model for echovirus 22 RNA composed of a linear RNA molecule with a 5' hairpin.     

Characterization of the Snow Mountain agent of viral gastroenteritis.

Snow Mountain agent (SMA) is a 27- to 32-nm virus which is the etiologic agent of outbreaks of acute gastroenteritis in Colorado and Vermont. SMA is morphologically similar to but antigenically distinct from the Norwalk and Hawaii agents of viral gastroenteritis but, like those agents, has not been cultivated in vitro. We purified and characterized SMA directly from human stool specimens containing the virus. The density of the SMA virion was 1.29 g/cm3 and 1.21 to 1.22 g/cm3 on potassium tartrate-glycerol gradients and 1.33 to 1.34 g/cm3 on cesium chloride gradients. SMA had an S value of 170 to 183S on a sucrose velocity gradient. The purified virion was iodinated, immunoprecipitated with acute and convalescent sera from volunteers challenged with SMA, and analyzed on polyacrylamide gels. The virion contains one major structural protein of 62,000 molecular weight, which is similar in size to the 59,000-molecular-weight protein found in the Norwalk virion. The biophysical properties and single structural protein of SMA most closely resemble those of the calicivirus group.     

Isolation and Characterization of a Herpesvirus from Leukemic Guinea Pigs

A guinea pig herpesvirus (GPHV) has consistently been isolated from leukemic lymphoblasts of strain-2 guinea pigs. GPHV is serologically related to the guinea pig herpes-like virus isolated by Hsiung and Kaplow. The virions of GPHV consist of an icosahedral capsid containing a dense nucleoprotein core enclosed in a double-layered membrane. The average diameters of GPHV virion and capsid were 166 and 101 nm, respectively. Studies on the morphogenesis of GPHV revealed that, as in other herpesvirus infections, only the naked capsids with or without the nucleoprotein core were found in the infected cell nuclei; it was also learned that the virion acquired its envelope by budding from the nuclear membrane of the infected cells. However, GPHV-infected cell nuclei also contained dense fibrous rods, resembling nucleo-protein core outside the capsids, and tubules resembling viral core protein. The capsids were often embedded in dense granular antigen. GPHV deoxyribonucleic acid (DNA) has a density of 1.716 g/ml in cesium chloride compared to herpes simplex virus DNA (rho = 1.728 g/ml) and cellular DNA (rho = 1.700 g/ml).     

Poliovirus and echovirus survival in Tetrahymena pyriformis culture in vivo

Axenic cultures of Tetrahymena pyriformis, strain I MT IV, grown in a defined medium at room temperature, were used to study interactions of these protozoa with vaccination strain L Sc 2ab of poliovirus type 1, vaccination strain P 712 of poliovirus type 2 and with type 30 echovirus, strain 480/78. T. pyriformis cultures in media containing 10(3.0) TCD50/1 ml of type poliovirus, 10(3.0) TCD50/1 ml of type 2 poliovirus or 10(2.5) TCD50/1 ml echovirus 30 and in virus-free medium did not differ one from another in their growth and die-away kinetics during the 21 days of observation. Two-day T. pyriformis cultures were infected with poliovirus 1 (initial concentration 10(3.2) TCD50/1 ml), and poliovirus 2 and echovirus 30 (initial concentrations 10(3.0) TCD50/1 ml). Viruses were titrated in test tube cultures of BGM cells. The supernatant fluid, standardized sediment and samples of control virus suspension free of protozoa were titrated after 0, 2, 6, 10, 13, 18, 28 and 30 days. Most of the virus in culture was found associated with the sediment, both in the period of active growth and during the die-away phase of T. pyriformis protozoa. The virus in sediment was present at higher titres and its survival time was longer than in virus in liquid phase. Thirteen days after the first contact between T. pyriformis and virus the sediment and supernatant fluid of the old protozoan culture and the T. pyriformis-free control viral suspension were taken and used as inocula for new two-day T. pyriformis cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical studies of respiratory syncytial virus. I. Density of respiratory syncytial virus and associated complement-fixing antigens in cesium chloride density gradient

Coates, Helen V. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Ben R. Forsyth, and R. M. Chanock. Biophysical studies of respiratory syncytial virus. I. Density of respiratory syncytial virus and associated complement-fixing antigens in a cesium chloride density gradient. J. Bacteriol. 91:1263-1269. 1966.-Concentrated fluids from respiratory syncytial (RS) virus-infected tissue cultures (HEp-2 and BEK) were subjected to equilibrium sedimentation in cesium chloride. When two antigenically distinct strains of RS virus (Long and 18537) were tested, approximately 90% of the infectious virus was recovered in a sharp, symmetrical peak with a density of 1.22 to 1.24. In a similar study, unconcentrated virus had a density of 1.25 to 1.27. Two immunologically distinguishable complement-fixing antigens (antigens A and B) were detected at densities of 1.28 to 1.32 and 1.23 to 1.37. In addition, the existence of a third antigen (density of 1.22 to 1.30) was suggested. The possible origin of these antigens is discussed relative to the known properties of RS virus and the other myxoviruses.     

Characterization of Hepatitis G Virus (GB-C Virus) Particles: Evidence for a Nucleocapsid and Expression of Sequences Upstream of the E1 Protein

Hepatitis G virus (HGV or GB-C virus) is a newly described virus that is closely related to hepatitis C virus (HCV). Based on sequence analysis and by evaluation of translational initiation codon preferences utilized during in vitro translation, HGV appears to have a truncated or absent core protein at the amino terminus of the HGV polyprotein. Consequently, the biophysical properties of HGV may be very different from those of HCV. To characterize HGV particle types, we evaluated plasma from chronically infected individuals with and without concomitant HCV infection by using sucrose gradient centrifugation, isopycnic banding in cesium chloride, and saline density flotation centrifugation. Similar to HCV, HGV particles included an extremely-low-density virion particle (1.07 to 1.09 g/ml) and a nucleocapsid of ~1.18 g/ml. One major difference between the particle types was that HGV was consistently more stable in cesium chloride than HCV. Plasma samples from chronically HGV-infected individuals and controls were assessed by a synthetic peptide-based immunoassay to determine if they contained HGV antibody specific for a conserved region in the coding region upstream of the E1 protein. Chronically HGV-infected individuals contained antibody to the HGV core protein peptide, whereas no binding to a hepatitis A virus peptide control was observed. Competitive inhibition of binding to the HGV peptide confirmed the specificity of the assay. These data indicate that HGV has a nucleocapsid and that at least part of the putative core region of HGV is expressed in vivo.     

Biophysical Properties of Frog Virus and Its Deoxyribonucleic Acid: Fate of Radioactive Virus in the Early Stage of Infection

Frog virus (FV-3) was banded by isopycnic centrifugation in cesium chloride, sucrose, or potassium tartrate. Two bands of infectivity were regularly found at positions in cesium chloride corresponding to densities of 1.26 and 1.30 g/cm(3), respectively. Deoxyribonucleic acid from either band had the following characteristics: double-stranded; a T(m) of 76.3 C in 0.1 SSC (0.015 m NaCl plus 0.015 m sodium citrate) and a buoyant density of 1.720 g/cm(3) in cesium chloride, corresponding to a guanine plus cytosine content of 56 to 58% and a molecular weight of 130 x 10(6) daltons, determined by velocity sedimentation. These data, together with electron micrographs of sections of cells infected with material from either band suggest that two types of infectious frog virus particles exists, rather than a second virus in the frog virus stocks. The composition of frog virus was determined. It was found that highly purified preparations of frog virus were composed of 55.8% protein, 30.1% deoxyribonucleic acid, and 14.2% lipid. The kinetics of adsorption and uncoating of FV-3 was studied with radioactive virus. Uncoating is comparatively rapid and in contrast to poxvirus is unaffected by inhibitors of protein synthesis.     

Density gradient centrifugation studies on rabies virus

Neurath, A. Robert (The Wistar Institute, Philadelphia, Pa.), Tadeusz J. Wiktor, and Hilary Koprowski. Density gradient centrifugation studies on rabies virus. J. Bacteriol. 92:102-106. 1966.-Cesium chloride density gradient centrifugation of rabies virus revealed a heterogeneous population of infectious virus particles, the majority of which showed a density of 1.20 g/ml. From results obtained by rate zonal centrifugation in preformed sucrose gradients, it was possible to calculate a sedimentation coefficient of about 600S for rabies virus. Sedimentation coefficients of about 23S and 10S were calculated for two soluble rabies antigens present in infected tissue-culture fluids, and they showed a density of 1.26 g/ml in cesium chloride solutions.     

Characterization of hog cholera virus. I. Determination of buoyant density

Horzinek, Marian (Tier?rztliche Hochschule, Hannover, West Germany). Characterization of hog cholera virus. I. Determination of buoyant density. J. Bacteriol. 92:1723-1726. 1966.-Hog cholera virus was subjected to cesium chloride density gradient centrifugation. Most of the infectious activity was detected in fractions with densities between 1.15 and 1.20 g/ml, with a peak at 1.16 g/ml. Infectivity was assayed by use of either the exaltation of Newcastle disease virus method or the hemagglutination exaltation and inhibition of cytopathic effect method.     

Characterization and classification of virus particles associated with hepatitis A. I. Size, density, and sedimentation.

Virus-like particles were purified from stools of patients in an epidemic of hepatitis A in Germany. When reference MS-1 chimpanzee pre-inoculation and convalescent sera were used, the close serological relationship of the purified particles to well-known isolates of hepatitis A could be established. On the other hand, the physicochemical characteristics of the particles were determined in parallel to the characteristics of a marker parvovirus (LuIII) and a marker picornavirus (poliovirus type 2). It could be shown that the majority of the hepatitis A-associated particles band at 1.34 g/ml in CsCl and, like poliovirus, sediment at about 160S. In addition, a distinct hepatitis A antigen was observed, which banded at 1.305 g/ml and sedimented between 50 and 90S. A further component accumulated in the density range of between 1.38 and 1.44 g/ml. However, it seemed to be rather labile. Upon reisolation from CsCl and sedimentation in sucrose, it resolved into a 160S, a 90 to 100S, and a 50S form. The size of the 160S particles (27 to 29 nm) could be readily distinguished from that of the parvovirus (22 to 24 nm). It is concluded, therefore, that hepatitis A-associated virus particles are more likely to be classified with the picornaviruses than with the parvoviruses.     

Chiral discrimination and antipicornavirus activity of 6-oxazolinylisoflavan

Racemic 6-oxazolinylisoflavan, a highly effective inhibitor of rhinovirus serotype 1B in vitro, was resolved by high-performance liquid chromatography on a chiral stationary phase in order to study the activity of the enantiomers against picornaviruses. The absolute configuration of the two isomers was determined by circular dichroism curves. The antipicornavirus activity of each isomer, separately collected, was evaluated in vitro against human rhinovirus serotype 1B, enterovirus 71, echovirus 6, coxsackievirus B4, and poliovirus type 2 by means of the plaque reduction assay. Both enantiomers were inhibitors of picornavirus replication with the degree of their activity depending on virus and isomer tested. © 1993 Wiley-Liss, Inc.     

Physical and Biological Properties of Dengue-2 Virus and Associated Antigens

Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients. Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface. Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm(3) and from cell culture at 1.24 g/cm(3). During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm(3). It was shown in electron micrographs to consist of virion fragments. A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm(3) in CsCl, and appeared as "doughnut" forms measuring 13.8 to 14 nm in diameter. A third component, with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.     

Defective Virions in Human Adenovirus Type 12

Purified preparations of human adenovirus type 12 showed two bands when subjected to isopycnic centrifugation in a density gradient of cesium chloride. Their density difference was about 0.003 g/ml, suggesting a small difference in their deoxyribonucleic acid to protein ratio. Virions with a lighter density can kill human KB cells and induce T antigen as efficiently as the heavy virions. However, they appeared incapable to form plaques. Two passages of the heavy infectious virions at low multiplicity of infection did not produce significant amounts of light virions; however, when it was passed at high multiplicity of infection, the light band became visible in a cesium chloride density gradient.     

Characterization of the Strain of Adenovirus Type 7 Carrying the Defective Monkey Cell-adapting Component

The strain of adenovirus type 7 carrying the defective monkey cell-adapting component (MAC) has been further characterized. MAC is more sensitive to inactivation by ultraviolet light than the associated adenovirus, which, in turn, is more rapidly inactivated than complete simian virus 40 (SV40). The 37% dose was 16 sec for MAC, 60 sec for adenovirus, and 84 sec for SV40. Filtration through membranes revealed that both MAC and adenovirus were retained by 100-mmu filters. MAC equilibrated in cesium chloride at the same buoyant density as the complete adenovirions (1.34 g/ml). Deoxyribonucleic acid extracted from green monkey kidney cells infected with MAC-adenovirus 7 did not hybridize in vitro with SV40 complementary ribonucleic acid. The two components (MAC and adenovirus) of the virus population possess such similar biophysical properties that their separation has not yet been achieved. Evidence to date indicates that MAC is probably not derived from SV40.     

Defective Virions of Reovirus

When purified preparations of stock reovirus, type 3, were digested with chymotrypsin, the virions were converted into two different types of particle. These new particles could be separated from each other by isopycnic centrifugation in cesium chloride gradients. One particle banded at a buoyant density of 1.43 g/cm(3), the other at a density of 1.415 g/cm(3). The former particle is termed the heavy (H) particle, the latter is the light (L) particle. The ratio of H/L particles varied between 0.5 and 0.25 in various purified preparations of virus. In electron micrographs, both H and L particles had the appearance and dimensions of viral cores. H particles were infectious for L cells. When plaques formed by stock virus, or by H particles, were picked and propagated in L cells, the majority of the clones gave rise only to H particles on chymotrypsin digestion. On continued serial passage of the clones, virions containing L particles again appeared in the progeny. The simplest explanation of these results was that stock virus was comprised of two populations of virions. One type of virion which contained H particles was infectious, whereas the other, which contained L particles, was not itself infectious and could replicate only in cells coinfected with an H particle virion. Added weight was given to this hypothesis by two observations. First, a small but definite separation of H and L virions could be achieved by isopycnic centrifugation in a gradient of cesium chloride. Second, L particles and virions containing L particles were both shown to lack the largest of the ten segments of double-stranded ribonucleic acid genome. Thus, L particle virions have defective genomes.     

Intracellular forms of simian virus 40 nucleoprotein complexes. II. Biochemical and electron microscopic analysis of simian virus 40 virion assembly.

The simian virus 40 virion assembly process was studied with pulse-labeling kinetics of virion proteins, CsCl gradient analysis, electron microscopy, and low-salt gel electrophoresis. The results obtained are consistent with the model of gradual addition and organization of capsid proteins around simian virus 40 chromatin. Empty virions, as observed in the CsCl gradient by previous workers, were found to be the dissociation product of immature virus. Histone H1 was found in simian virus 40 chromatin and virion assembly intermediates but not in the mature virion banding at 1.34 g/ml in the CsCl gradient.     

Polyadenylate sequences of human rhinovirus and poliovirus RNA and cordycepin sensitivity of virus replication.

The polyadenylate [poly(A)] content of the genome RNA of human rhinovirus type 14 (HRV-14) is nearly twice as large as that of the genome RNA of poliovirus type 2. The poly(A) content of viral RNA was determined to be the RNase-resistant fraction of 32P-labeled viral RNA extracted from purified virions. Polyacrylamide gel electrophoresis indicated that the poly(A) sequences of HRV-14 are more heterogenous and on an average larger than those of poliovirus RNA. On the basis of susceptibility to micrococcal polynucleotide phosphorylase the rhinovirus genome terminates in poly(A). Replication of both viruses is almost totally inhibited by cordycepin at 50 mug/ml. At lower concentrations, rhinovirus replication is more sensitive to cordycepin than poliovirus replication. Addition of cordycepin (75 mug/ml) to infected culture prior to or during viral RNA replication results in more or less complete inhibition of virus-specific RNA synthesis. The results do not indicate that cordycepin sensitivity of either virus is due to preferential inhibition of viral poly(A) synthesis by this antibiotic.     

Structure of Infectious Bursal Disease Virus

Infectious bursal disease virus of chickens was purified, and its structure was examined by the negative-staining technique in the electron microscope. The buoyant density of infectious bursal disease virus in CsCl was found to be 1.34 g/cm(3). The morphological details suggest that the capsid of the virion consists of a single layer of 32 capsomeres arranged in 5:3:2 symmetry. The virion measured about 55 nm in diameter and had no envelope.