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.     

Isolation and Characterization of Kinetoplast DNA Networks and Minicircles from Crithidia fasciculata*

SYNOPSIS. Covalently closed kinetoplast DNA networks have been isolated from stationary phase Crithidia fasciculata cells by a technic involving selective pelleting of the networks at a low centrifugal field. Approximately 62% of the kinetoplast DNA of the cell was recovered free of nuclear DNA by simple differential centrifugation. Purified kinetoplast DNA networks were visualized both in the electron microscope and in the light microscope. Closed networks sedimented as a homogeneous band both in neutral and alkaline sucrose, with an s_20w in neutral sucrose of approximately 5 × 10³. Closed monomeric minicircles were isolated from purified networks by mild sonication and band sedimentation in alkaline sucrose. Several physical properties of closed monomeric minicircles were measured. These included molecular weight, buoyant density in CsCl, superhelix density and sedimentation coefficient.     

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.     

兔的一种新病毒：Ⅱ.一株兔出血症...

In this paper a strain of Rabbit Hemorrhagic Disease Virus (RHDV) was isolated and purified from the diseased rabbit livers with a method of using chloroform, two-phase of polyethylene-glycol-dextran sulfate sodium and sucrose density gradient centrifugation. Purified virus was nonenveloped, icosahedeal symmetry with a triangulation number of 3, and 33-37 nm in diameter. The capsid was composed of 32 capsomeres with central holes in an outer diameter of about 9nm. Two types of viral particles having different sedimentation coefficient, 130s and 166s could be identified after sucrose density gradient centrifugation. Probably no less than four virion proteins with molecular weight of 66.4, 65.0, 63.5, 41.0 x 10(3) dalton were detected by SDS-polyacrylamide gel electrophoresis. Viral nucleic acid was extracted from purified virus by using SDS-proteinase K-phenol. Tests with diphenylamine, formaldehyde, and staining with acridine orange as well as the curves of thermal denaturation showed that this kind of virus had a single-stranded DNA. The molecular weight of the ssDNA was approx 2.1 x 10(6) dalton as determined by electron microscopy. Data indicate that the RHDV may like the parvovirus of the family Parvoviridae.     

Size and Composition of Marek''s Disease Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified nucleocapsids of Marek's disease herpesvirus (MDV) was cosedimented with T4 and with herpes simplex virus (HSV) DNA in neutral sucrose density gradients and with T4 DNA in alkaline sucrose density gradients. These experiments indicated that the intact MDV DNA had a sedimentation constant of 56S corresponding to a molecular weight of 1.2 x 10(8) daltons. In the alkaline gradients, the largest and most prominent band contains a DNA sedimenting at 70S corresponding to 6.0 x 10(7) daltons in molecular weight. The DNA is therefore double-stranded and not cross-linked. Isopycnic sedimentation of the MDV DNA molecules with SPO1, Micrococcus lysodeikticus, and HSV DNA gave a density of 1.705 g/cm(3) corresponding to 46 guanine plus cytosine moles per cent. Lastly, in hybridization tests the DNA hybridized with RNA of infected cells but not with that of uninfected cells supporting the conclusion that it is viral.     

Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA.

Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.     

Morphology of a Virus of Blue-green Algae and Properties of Its Deoxyribonucleic Acid

The morphology of Safferman's virus of blue-green algae (phycovirus LPP-1) has been studied by electron microscopy and physicochemical methods. The virion has a short (100 to 200 A long, 150 A in diameter) forked tail, with an outer sheath, an inner core, and a capital attached to one of the vertices of a polyhedral head. The head capsid edge-to-edge distance is 600 A, based upon internal calibration of the magnification in electron micrographs by use of the line-line spacing of catalase crystals. Measurements of absorbancy and infectivity, and electron microscopy across the band of virus after zone centrifugation on a sucrose gradient, indicated that infectivity was correlated with the short-tailed particles described. The viral deoxyribonucleic acid (DNA) is linear, with a contour length of 13.2 +/- 0.5 mu, measured by the Kleinschmidt method. Its sedimentation coefficient, S(0) (20, w), is 33.4 +/- 0.7 S. These values are consistent with a molecular weight of 27 x 10(6) for the viral DNA. Based upon buoyant density in CsCl and thermal denaturation, the guanine-cytosine content of the DNA is 53%. The viral DNA was used as template for in vitro ribonucleic acid (RNA) synthesis by Escherichia coli RNA polymerase. This RNA annealed to 18% of the sequences in the viral DNA, 0.5% of the sequences in bacteriophage T7 DNA, and 0.25% of the sequences in Plectonema boryanum DNA, at saturating levels of RNA in the Hall-Nygaard hybridization assay. The lack of homology with T7 DNA is of interest because the two viruses are very similar morphologically. The lack of homology with host DNA suggests that this algal virus is a poor candidate for transduction.     

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.     

Structure and synthesis of a lipid-containing bacteriophage. The molecular weight and other physical properties of bacterophage PM2.

Several physical and chemical parameters of bacteriophage PM2 have been measured. The sedimentation constant was determined to be s-20,w=293 S. The buoyant density in sucrose at 20 degrees C was 1.24 g cm+-3 and in CsCl at 25 degrees C was 1.29 g cm-3. The high-speed equilibrium centrifugation method of Yphantis (1964) was used to measure the molecular weight of PM2. The necessary auxiliary parameters were also determined. A value of 0.771 plus or minus 0.005 cm-3 g-1 for the apparent specific volume at constant chemical potential in 1 M sodiium chloride has been obtained by pycnometry; the viral concentration was determined using the absorption coefficient at 260 nm (4.60 plus or minus 0.10 cm-2 mg-1), which in turn was calculated from the phosphorous content of the virus (17.89 plus or minus 0.28 mu-g of P per mg dry weight dry weight of virus). The molecular weight of PM2 determined with these parameters is (44.1 plus or minus 1.2 x 10-6). From the phosphorous content of the virus, the percentage of phosphorous known to be in its DNA (Camerini-Otero and Franklin, 1972), and the molecular weight of the bacteriophage, we have calculated a molecular weight for PM2 DNA of 6.26 x 10-6, which confirms values determined using empirical relationships.     

Further characterization of intracytoplasmic A particle-associated DNA.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.     

Purification and Biophysical Properties of Acute Haemorrhagic Conjunctivitis Virus

The buoyant density of acute haemorrhagic conjunctivitis virions labeled with either [(3)H]uridine or [(3)H]leucine was 1.34 g/ml in CsCl and 1.25 g/ml in sucrose. RNA extracted from the virions gave a sedimentation coefficient of approximately 34S in sucrose, and was found to be sensitive to RNase. Molecular weight of RNA was calculated to be 2.5 x 10(6) using poliovirus RNA for reference.     

Some Properties of the PBP1 Transduction System in Bacillus pumilus

Bacteriophage PBP1 is a flagella-specific virus that performs generalized transduction in strains of Bacillus pumilus. PBP1 is morphologically and serologically distinct from two other flagella-specific phages, PBS1 and SP-15, which perform generalized transduction in certain Bacillus species. The DNA extracted from PBP1 particles has a buoyant density of 1.690 g/cm(3) in cesium chloride gradients, a melting temperature of 86.1 C, and a sedimentation velocity of 47S in neutral sucrose gradients. Assuming the molecule is a linear duplex, PBP1 DNA has a molecular weight of approximately 76 x 10(6). In two strains of B. pumilus which are sensitive to both PBP1 and PBS1, co-transducible genetic markers are more tightly linked by PBS1 transduction than by PBP1 transduction. The size of the fragment of bacterial DNA carried by PBP1-transducing particles, inferred from transduction studies and sedimentation analysis of viral DNA, suggests that PBP1 may be useful for genetic studies of extrachromosomal DNA elements present in two strains of B. pumilus. Genetic exchange of chromosomally located genes between the plasmid(+) and plasmid(-)B. pumilus strains NRS 576 and NRRL B-3275 has been demonstrated by PBP1 transduction.     

Defective Friend Spleen Focus-Forming Virus: Interfering Properties and Isolation Free from Standard Leukemia-Inducing Helper Virus

Defective Friend spleen focus-forming virus (SFFV) is able to interfere with the ability of its naturally occurring leukemia-inducing helper virus (LLV-F) to induce XC plaque formation in several different strains of mouse embryo cells. This interference has been observed by using two different SFFV preparations, one contained in an NB-tropic stock of Friend virus (FV) complex, and the second present in a C57BL-adapted strain of FV complex containing an associated B-tropic LLV-F helper. The LLV-F in NB-tropic FV complex effectively induced XC plaques in C57BL/6 (Fv-1^bb; Fv-2^rr) mouse embryo fibroblasts (MEF) only in the absence of coinfecting SFFV, indicating that Fv-2-associated resistance to SFFV-induced focus formation in vivo does not necessarily extend to the restriction of SFFV function(s) in vitro (i.e., in Fv-2^rr C57BL MEF). SFFV interference appears to be an intracellular event since LLV-F can adsorb onto, penetrate, and rescue defective murine sarcoma virus (MSV) from transformed 3T3FL S⁺L⁻ cells with equal efficiency in the presence and absence of SFFV. However, significantly fewer LLV-infected S⁺L⁻ cells released LLV-F progeny if SFFV was present. These observations suggest that Friend SFFV may be classified as a defective, interfering (DI) particle. Further support for this conclusion has come from studies designed to investigate two physical properties of defective SFFV particles. SFFV layered onto a 0 to 20% sucrose sedimentation gradient was recovered as a symmetrical band of virus that sedimented more slowly than standard LLV-F particles. Pooled SFFV-containing gradient samples contained visualizable type C virus particles and occasionally small amounts of detectable LLV-F. In an attempt to determine the buoyant density of sedimentation gradient-purified SFFV, pooled SFFV samples were layered onto a 25 to 50% sucrose equilibrium density gradient and were centrifuged to equilibrium. Greater than 50% of the infectious SFFV originally layered onto this gradient was recovered and seen as a narrow symmetrical band with peak SFFV infectivity at a sucrose density of 1.14 g/ml. The observed difference between SFFV and LLV-F buoyant densities appears to be related to an inherent physical property of each virus. Mixtures of these two viruses express the buoyant density of that virus population which is in excess in fabricated FV complexes probably due to the formation of SFFV-LLV aggregates. Finally, gradient-purified SFFV failed to induce XC plaques in MEF and did not function to rescue MSV as expected since SFFV itself is replication defective.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

Physical properties of plasmid Mor174, which determines bacteriocin production in Proteus morganii 174.

Plasmid Mor174 has a molecular weight of 3.6 X 10(6) and a buoyant density of 1.6994 g/cm3. The covalently closed circular form has a sedimentation coefficient of 22S. These are 30 to 40 plasmid copies per genome equivalent, but growth in chloramphenicol results in amplification of the copy number to 600. In Proteus morganii 174, Mor174 coexists with a cryptic plasmid of molecular weight 15.8 X 10(6) and a buoyant density of 1.7170 g/cm3.     

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.     

Role of lysine in the replication of reovirus: I. Synthesis of complete and empty virions

Lysine is essential for the replication of infectious reovirus. Omission of lysine from the extracellular medium not only permitted the continued synthesis of structural viral proteins and viral double-stranded ribonucleic acid (RNA), but also caused an enhanced formation of viral structures which were separable by isopycnic sedimentation of CsCl into a top band consisting of empty particles with a buoyant density of 1.29 g/cm³ and essentially free of viral RNA, and two lower bands which were difficult to resolve and had an average buoyant density of 1.37 g/cm³. The lower bands contained most of the viral nucleic acid. The above effects were reversed when lysine was restored early after infection. In contrast, a single band with a buoyant density of 1.38 g/cm³ was obtained from lysine-plus infected cells.     

Properties of Johnsongrass Chlorotic Stripe Mosaic Virus

Physicochemical, biological, and cytopathological properties of Johnsongrass chlorotic stripe mosaic virus (JCSMV) found in Iran were investigated. Virus particles were polyhedral, showed a knobbed surface structure, were c. 30 nm in diameter and had a buoyant density of 1. 359 g/ml in cesium chloride. Virions contained one major protein with a molecular weight of 41 kd and a single species of ssRNA with a molecular weight of 1. 43 × 10⁶ d. Acid hydrolysis of the virus followed by thin-layer electrophoresis gave the following molar percentages of the bases: A: 23. 5, G: 27. 5, C: 26 and U: 23. Separation of nucleotides of the viral RNA using alkaline hydrolysis was not successful. Mechanical inoculation of freshly purified virus or isolated RNA failed to infect Johnsongrass or maize plants. The virus was readily detected by ELISA in seeds from infected plants and young seedlings raised from such seeds, but not in later stages of growth. Ultrathin sections of infected cells showed high concentrations of virus particles in the cytoplasm and vacuoles. Virus-like particles also occurred in the stroma of chloroplasts and mitochondria. Cisternae of endoplasmic reticulum (ER) were often extremely inflated and filled by a fine fibrillar material. Small membrane-associated vesicles were frequently found in ER elements and occurred also in the permuclear space. Based on particle structure, properties of the nucleic acid, molecular weight of the coat protein and cytopathology, the virus resembles carmoviruses. However, lack of mechanical transmissibility is not known for any virus classified with this group. No serological reaction was detected with a total of 30 antisera to carmoviruses and other isometric viruses.     

Sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins

The sedimentation behavior of transmissible gastroenteritis coronavirus (TGEV) was analyzed. Upon sucrose gradient centrifugation, the major virus band was found at a density of 1.20 to 1.22 g/cm(3). This high density was observed only when TGEV with a functional sialic acid binding activity was analyzed. Mutants of TGEV that lacked sialic acid binding activity due to a point mutation in the sialic acid binding site of the S protein were mainly recovered at a lower-density position on the sucrose gradient (1.18 to 1.19 g/cm(3)). Neuraminidase treatment of purified virions resulted in a shift of the sedimentation value from the higher to the lower density. These results suggest that binding of sialoglycoproteins to the virion surface is responsible for the sedimentation behavior of TGEV. When purified virions were treated with octylglucoside to solubilize viral glycoproteins, ultracentrifugation resulted in sedimentation of the S protein of TGEV. However, when neuraminidase-treated virions or mutants with a defective sialic acid binding activity were analyzed, the S protein remained in the supernatant rather than in the pellet fraction. These results indicate that the interaction of the surface protein S with sialoglycoconjugates is maintained after solubilization of this viral glycoprotein by detergent treatment.