Characterization and classification of virus particles associated with hepatitis A. II. Type and configuration of nucleic acid.

Virus particles banding at 1.34 g/ml in CsCl and sedimenting at 160S in sucrose gradients were isolated from fecal specimens of patients suffering from hepatitis. In the presence of 4 M urea and about 90% formamide, these particles released linear nucleic acid molecules of the kinked appearance characteristic of single-stranded RNA or single-stranded DNA. They could be distinguished from the nucleic acid of phage lambda added to the preparation as a marker for double-stranded configuration. Experiments in which the virus particles under investigation were incubated at pH 12.9 at 50 degrees C for 30 min revealed that their nucleic acid molecules were hydrolyzed as readily as the RNA genome of poliovirus type 2 analyzed in parallel. Both the single-stranded DNA of phage phiX174 and that of parvovirus LuIII, however, proved unaffected by this treatment, and the double-stranded DNA of phage lambda was denatured to single-stranded molecules. It was concluded, therefore, that the virus of human hepatitis A contains a linear genome of single-stranded RNA and has to be classified with the picornaviruses.     

Characterization and classification of virus particles associated with hepatitis A. III. Structural proteins

Hepatitis A virus was purified from fecal samples collected at various times in the incubation period of patients with naturally acquired hepatitis A. The proteins of particles banding at around 1.34 g/ml in CsCl and sedimenting at about 160S were radioiodinated in vitro and separated by electrophoresis on polyacrylamide gels in the presence of 0.1% sodium dodecyl sulfate and 8 M urea. Under these conditions, the capsid proteins resolved into four polypeptides with molecular weights of approximately 31,000, 24,500, 21,000, and 9,000, respectively. A fifth protein of about 40,000 daltons in size and assumed to be equivalent to the precursor polypeptide VP0 of the picornaviruses was present in particles sedimenting at only 150 to 155S and banding at around 1.33 g/ml in CsCl. The physicochemical characteristics of these particles are consistent with those of the provirion structures of picornaviruses. In several of the fecal samples, these particles represented a considerable fraction of all particles present. The significance of this finding with respect to the antigenicity of hepatitis A antigen extracted from stool specimens is discussed.     

Buoyant Density of the Hepatitis A Virus-Like Particle in Cesium Chloride

We recently visualized by immune electron microscopy a virus-like particle in the stools of patients with hepatitis A. The particle measured approximately 27 nm in diameter and morphologically resembled a picornavirus or parvovirus. To further characterize this particle, we have determined its buoyant density in cesium chloride (CsCl) by ultracentrifugation. Hepatitis A particles from three positive stool specimens were isopycnically banded in separate experiments, and the gradient fractions were examined for particles by immune electron microscopy by using hepatitis A convalescent sera. In each experiment, the particles were observed in a normal distribution about a peak fraction with a mean density of approximately 1.4 g/cm(3). The buoyant density of 1.4 g/cm(3) in CsCl together with its morphology and the reported resistance of hepatitis virus to acid, ether, and heat suggest that this particle is parvovirus-like.     

Characterization of Aleutian disease virus as a parvovirus.

We characterized a strain of Aleutian disease virus adapted to growth in Crandall feline kidney cells at 31.8 degrees C. When purified from infected cells, Aleutian disease virus had a density in CsCl of 1.42 to 1.44 g/ml and was 24 to 26 nm in diameter. [3H]thymidine could be incorporated into the viral genome, and the viral DNA was then studied. In alkaline sucrose gradients, Aleutian disease virus DNA was a single species that cosedimented at 15.5S with single-stranded DNA from adeno-associated virus. When the DNA was analyzed on neutral sucrose gradients, a single species was again observed, which sedimented at 21S and was clearly distinct from 16S duplex adeno-associated virus DNA. A similar result was obtained even after incubation under annealing conditions, implying that the bulk of Aleutian disease virus virions contained a single non-complementary strand with a molecular weight of about 1.4 X 10(6). In addition, two major virus-associated polypeptides with molecular weights of 89,100 and 77,600 were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virus purified from infected cultures labeled with [35S]methionine. These data suggest that Aleutian disease virus is a nondefective parvovirus.     

Biological activity and electron microscopy of poliovirus 14S particles obtained from alkali-dissociated procapsids.

Highly purified 14S subunit particles were obtained from alkali-dissociated poliovirus type 1 procapsids (naturally occurring empty capsids in poliovirus-infected cells) to compare their morphological and biophysical properties with those of naturally occurring 14S particles. Procapsid-derived 14S particles (PC-14S), like naturally occurring 14S particles, were capable of self-assembly into an empty shell in buffer or extracts from uninfected cells. These empty capsids always exhibited pIs more acidic than those of procapsids but were themselves distinguishable by their respective pIs. Nevertheless, if PC-14S or naturally occurring 14S particles were incubated with extracts made from poliovirus-infected cells, procapsidlike empty shells were formed. This clearly showed that the 14S particle, however obtained, possesses the information to form an empty shell of correct dimensions but of improper conformation, unless a factor present in poliovirus-infected cells is present. With the electron microscope, the PC-14S subunit frequently was seen as a pentagonal structure with a diameter of 20.4 +/- 1.4 nm, a size somewhat larger than expected for a subunit composing 1/12th of the poliovirus surface. Upon self-assembly in vitro, the empty shell formed exhibited a diameter of 29 +/- 1 nm and a wall thickness of ca. 6 to 7 nm. It was necessary to avoid CsCl banding of procapsids in their preparation as this treatment altered both their pI and their sensitivity to alkali dissociation into 14S subunits. The relevance of these findings to the nature and role of procapsids and the requirement for a morphopoietic factor in poliovirus morphogenesis is discussed.     

小鹅瘟病毒纯化及其理化特性的研究

详细描述了小鹅瘟病毒(Goslin—plague virus,GPV)扬州株的浓缩和纯化过程,并论述了其理化特性。试验证明,GPV在氯化铯中主要病毒区带的浮密度为11.31～1.35g/ml,电镜下可见空壳和实心两种病毒粒子,大小20～22nm,沉降系数90.5S。用Sepharose 4B柱层析纯化的病毒,等电点为4.3。GPV有3种结构多肽,即Vp1、Vp2和Vp3,分子量分别为85 000,61 000,57 500道尔顿,其中Vp3为主要结构多肽,纯化的病毒粒子能使鹅胚致死。     

Virus-like particles from killer, neutral, and sensitive strains of Saccharomyces cerevisiae.

Procedures were developed for purification of virus-like particles (VLPs) from killer, neutral, and sensitive strains of Saccharomyces cerevisiae. Morphologically similar spherical VLPs measuring 40 nm in diameter were extracted from all three phenotypes. The particles were partially purified by high-speed centrifugation through a layer of CsCl (1.26 g/cm3) and sucrose density gradient centrifugation. Gradient-purified preparations contained three centrifugal species that sedimented at approximately 43, 102, and 162S. The 43S component is considered to be an artifact. Preparations from killer strains contained three double-stranded RNA (ds-RNA) components with molecular weights of 1.19 x 10(6), 1.29 x 10(6) and 2.54 x 10(6). VLPs from neutral and sensitive strains contained only the largest ds-RNA species. VLP preparations were subsequently separated into two major density components by CsCl equilibrium gradient centrifugation. The light component banding at 1.28 to 1.30 g/cm3 was void of nucleic acid, and the heavy component banding at 1.40 g/cm3 contained only the largest ds-RNA species.     

Physical, chemical and morphologic dimensions of human hepatitis A virus strain CR326 (38578).

CR326 human hepatitis A virus purified by isopycnic banding from infected marmoset sera was shown to consist of 27 nm spherical particles on electron microscopic examination. The particles were identified as hepatitis A virus by tests for infectivity and by specific neutralization of infectivity with convalescent human hepatitis A serum. Also, indentical 27 nm viruses in liver extracts gave specific reactions with hepatitis A antisera when tested by immune electron microscopy. The bouyant density of the virus in CsCl was 1.34 and it was heat (60 degrees), ether and acid stable but was destroyed by heat (100 degrees), formalin (1:4000) and ultraviolet irradiation. Electron microscopic studies of sections of infected marmoset liver showed intracytoplasmic virus particles, usually in vesicles. Presumptive findings for RNA, together with the intracytoplasmic location of the virus, indicated the virus to be of RNA-type. The attributes of the virus indicate it is closely related to the enterovirus family and not to hepatitis B virus.     

Immunological and biophysical alteration of hepatitis B virus antigens by sodium hypochlorite disinfection.

Sodium hypochlorite (NaOCl) was examined as an effective disinfectant in hepatitis laboratories. Concentrations of NaOCl containing 5,600 ppm (5,600 microgram/ml) of available chlorine were found to be effective in destroying the antigenicity of hepatitis B surface antigen (HBsAg) in virion-rich plasma after an exposure time of 1 min or more. In the treatment of protein-deficient solutions containing HBsAg, smaller concentrations of available chlorine (less than 500 pm) are equally effective. Neither 17-to 25-nm HBsAg particles nor 45-nm virion particles could be detected by electron microscopy after treatment. chemical interaction of protein and NaOCl was confirmed by isoelectrofocusing of 125I-labeled HBsAg. More than 90% of the labeled material was found at pH 3.0 or lower, indicating complete antigen oxidation. Labeled HBsAg was reduced in density from 1.21 g/cm3 in CsCl to approximately 1.07 g/cm3 after treatment with NaOCl. Both hepatitis B core antigen and deoxyribonucleic acid polymerase activity were significantly reduced after interaction with hypochlorite solutions. These results show that NaOCl destroys hepatitis B antigenicity and virus structures and therefore may be utilized as a disinfectant for the virus.     

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.     

Proteinaceous Virus-like Particles from an Isolate of Aspergillus flavus

Virus-like particles were purified from a single nonaflatoxin-producing isolate of Aspergillus flavus. The virus-like particles were spherical, measuring 27 to 30 nm in diameter, were electrophoretically homogeneous, and sedimented at approximately 49S. The particles had a buoyant density of 1.28 g/cm(3) in CsCl and contained no detectable nucleic acid.     

Purification of hepatitis A virus from chimpanzee stools.

Hepatitis A virus antigen was purified from early acute-phase chimpanzee stools by a rapid three-step procedure using 7% polyethylene glycol precipitation, CsCl banding, and Sepharose 2B column chromatography. Electron microscopic examination of the hepatitis A virus entigen preparation revealed highly purified hepatitis A virus particles.     

Mechanical Transmission, Purification and Properties of an Isolate of Maize Stripe Virus from Venezuela

A Venezuelan isolate of maize stripe virus (MStpV) was successfully transmitted mechanically and by the leafhopper Peregrinus maidis from field infected plants to sweet cv. Iochief. After purification of maize infected with MStpV, fine spiral filamentous particles about 4 nm in diameter and with variable lengths were consistently associated with a nucleoprotein band present in CsCl or Cs₂SO₄ isopycnic gradients. Purified preparations exhibited a typical nucleoprotein absorption spectrum with a maximum at 260–263 nm and a minimum at 240–243 nm and a 260–280 ratio of 1.38. The density of the nucleoprotein in CsCl gradients was estimated at 1.29 g/ml. The sedimentation coefficient was calculated at 62 S. The nucleoprotein consisted of 5 % single stranded RNA and a capsid protein of molecular weight 33.500 daltons. Large quantities of non-capsid proteins were isolated from infected tissue with a molecular weight of 17.500 and 16.500 daltons. Peregrinus maidis, injected with purified MStpV preparation failed to transmit the disease to healthy plants. However, they were infectious when injected with clarified infected plant sap. Antisera against capsid and non-capsid proteins from MStpV-Florida strain reacted positively with the Venezuelan antigens.     

Purification of infectious pancreatic necrosis (IPN) virus and comparison of polypeptide composition of different isolates.

Infectious pancreatic necrosis (IPN) virus was partially purified by freon extraction of infected CHSE-214 cells and concentrated by polyethylene glycol (PEG) precipitation of virus from the medium. Both methods resulted in virus concentrates that could be further purified by two CsCl gradient centrifugations with little loss of infectivity. A Recovery of 80 to 100% of the virus infectivity was obtained and over 100-fold concentration of viral infectivity was achieved by these methods. This purification was used to compare 10 isolates of IPN virus with regard to their physiochemical properties by electron microscopy, buoyant density in CsCl, and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified virions. Electron-microscopic observations showed that the virus isolates were identical in that they were isometric, hexagonal in profile, and had a particle diameter of 71 nm. The buoyant densities of the virus isolates in CsCl were found to be 1.33 g/ml. SDS-gel electrophoresis of the virus isolates revealed the presence of three polypeptides of molecular weight 50, 30, and 27 x 10(3) designated as VP50, VP30, and VP27, respectively.     

Incomplete and Aberrant Particles of Actinophage MSP2 from Lysates of Streptomyces venezuelae

Lysates of actinophage MSP2, propagated on Streptomyces venezuelae S13, contain at least 10(11) PFU/ml. During purification by centrifugation methods and by adsorption chromatography, a number of types of aberrant and incomplete phage particles were seen by electron microscopy. Infectious MSP2 had a buoyant density in CsCl of 1.52 g/cm(3) and an absorbance at 260 nm relative to that at 280 nm (A(260)/A(280)) of 1.53. Empty capsids banded at 1.276 g/cm(3) and partially filled capsids banded at 1.351 g/cm(3), and A(260)/A(280) ratios were 0.77 and 1.24, respectively. Two kinds of light capsids found in CsCl fractions of 1.278 g/cm(3) probably include the 1.276 component. Some capsids were joined by tail-like structures. Ghosts and polyheads also were present. Aberrant particles observed by electron microscopy included two-tailed actinophage, phage with abnormal tail positions, and large-headed phage.     

Structural relationships between the surface antigens of ground squirrel hepatitis virus and human hepatitis B virus.

Several physical, chemical, and serological properties of surface antigen particles from ground squirrel hepatitis virus (GSHsAg) and human hepatitis B virus (HBsAg) were compared. GSHsAg and HBsAg particles were purified from positive sera by gel chromatography and isopycnic centrifugation. Both antigens consisted mainly of spherical particles with an average diameter of approximately 20 nm and a buoyant density in CsCl of approximately 1.19 g/ml. Their UV absorption spectra indicated the presence of more tryptophane than tyrosine and the absence of detectable nucleic acid. GSHsAg was found to contain two major polypeptides of approximately 23,000 and 27,000 daltons, with electrophoretic migration rates distinctly faster than those of the two major polypeptides of HBsAg particles. After radiolabeling of purified antigen preparations with Bolton-Hunter reagent, the two major polypeptides of GSHsAg showed almost identical tryptic peptide maps. The tryptic peptide map of the major polypeptide from GSHsAg contained 13 of 37 spots also present in the map of the major HBsAg polypeptide, and 13 of 27 spots in the map of the major HBsAg polypeptide were also present in the map of the major GSHsAg polypeptide. This suggests considerable sequence homology between the major surface antigen polypeptides of the two viruses. However, there was only a weak serological cross-reactivity between antigens of the two viruses. Using an anti-HBs-containing serum with a relatively strong cross-reactivity, GSHsAg was found to consist of at least two antigenically different subspecies. The more strongly cross-reacting from had a slightly higher buoyant density than the other antigenic form.     

RNA binding properties of poliovirus subviral particles.

The mechanism of encapsidation of the RNA genome of poliovirus and other picornaviruses is unknown. To test whether any of the putative assembly intermediates of poliovirus could interact directly with the poliovirus RNA genome, poliovirus RNA was attached to magnetic streptavidin beads and incubated with partially purified extracts containing 35S-labeled 14S pentamer and 75S empty-capsid subviral particles from infected cells. The amount of labeled protein bound to the beads was monitored, thus testing the RNA-binding activities of only the labeled viral proteins in the preparations. In this assay, nonspecific RNA-binding activity was displayed by the 14S pentameric particles and mature virons. 75S empty capsids displayed no propensity to associate with RNA. 14S pentamers were demonstrated to form rapidly sedimenting complexes and to undergo a conformational alteration upon RNA binding. These findings are consistent with a direct role for the 14S pentameric particles in RNA packaging during poliovirus morphogenesis.     

罗氏沼虾体内两种病毒颗粒的分离、纯化与核酸特性

从患肌肉白浊症状的罗氏沼虾幼苗体内分离纯化得到两种大小不同的病毒颗粒.这两种病毒颗粒均为对称的20面体结构,表面光滑,无囊膜,对氯仿不敏感.一种是直径为26nm～27nm的颗粒,在氯化铯中的密度为132g/cm3,病毒基因组含两段单链的RNA,分别为30kb和12kb,具有诺达病毒科成员的特征.一种是直径为14nm～16nm的颗粒,在氯化铯中的密度为133g/cm3,含有一段大小为09kb的单链RNA,拟为卫星病毒样颗粒或辅助病毒.     

Characterization of the ion channels formed by poliovirus in planar lipid membranes.

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.