Frederick L. Schaffer

Survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids.

SummaryInfluenza A virus, strain WSNH, propagated in bovine, human and chick embryo cell cultures and aerosolized from the cell culture medium, was maximally stable at low relative humidity (RH), minimally stable at mid-range RH, and moderately stable at high RH. Most lots of WSNH virus propagated in embryonated eggs and aerosolized from the allantoic fluid were also least stable at mid-range RH, but two preparations after multiple serial passage in eggs showed equal stability at mid-range and higher RHs. Airborne stability varied from preparation to preparation of virus propagated both in cell culture and embryonated eggs. There was no apparent correlation between airborne stability and protein content of spray fluid above 0.1 mg/ml, but one preparation of lesser protein concentration was extremely unstable at 50 to 80 per cent RH. Polyhydroxy compounds exerted a protective effect on airborne stability.

Purification of Poliomyelitis Viruses propagated in Tissue Culture.

Abstract A procedure was developed for the purification and concentration of Mahoney, MEF-1, and Saukett strains of poliomyelitis virus from large volumes of tissue culture fluid. The basic procedure involved precipitation, butanol extraction, ultracentrifugation, and treatment with nucleases. Final separation of virus particles from residual cellular contaminants was achieved by electrophoresis or sedimentation in a density gradient. From tissue culture fluid in which the virus represented approximately 0.1% of the total protein, preparations of virus particles, some of which were crystalline, were obtained which closely approached chemical purity. Such preparations make possible studies of the serological, chemical, and physical properties of the virus.

The ultramicrodetermination of glycogen in liver; a comparison of the anthrone and reducing-sugar methods.

Summary 1. Procedures are described for determining glycogen in liver samples sufficiently small that a biopsy specimen may also provide material for other analyses. 2. Conditions for the colorimetric determination with anthrone reagent were modified to be applicable to quantities as low as 1 μg. (as glucose). 3. The microgram reducing-sugar method using ferricyanide oxidation and ceric sulfate titration was simplified and compared with the anthrone method. 4. Evidence is presented to show that glycogen does not vary greatly from site to site within one lobe of liver.

Purification and Properties of Poliovirus

Publisher Summary This chapter attempts to purify, identify, and characterize poliovirus particles with respect to their physical, chemical, and serological properties. Polioviruses of human origin are considered that are classified immunologically as Types 1, 2, and 3. Occasional reference is also made to the FA strain of mouse encephalomyelitis virus that is not classified as a poliovirus, although it shows some resemblance to murine poliovirus, Type TO. The purification procedures employed have been those found applicable to macromolecular proteins and enzymes in general. They include both physical and chemical techniques, such as ultrafiltration, ultracentrifugation, adsorption and elution, precipitation by salts or alcohols, as well as digestion of protein, and nucleic acid contaminants with enzymes. The identification of a virus particle requires the establishment of a relationship between infectivity and an adequately described physical particle. With polioviruses, this relationship has been demonstrated for a spherical particle of 27-30 mμ diameters. There is some suggestion that poliovirus infectivity can also be associated with a smaller particle. Identification and purification of poliovirus particles have made it possible to observe their physical properties directly, and thereby supplement the early, limited knowledge of their properties from indirect measurements.

Binding of proflavine by and photoinactivation of poliovirus propagated in the presence of the dye.

Abstract Proflavine was bound within poliovirus particles, presumably to the ribonucleic acid, when the dye was present up to concentrations of 5 μg/ml in the tissue culture medium in which the virus was propagated. The quantity of dye bound was dependent upon the concentration in the medium, reaching a maximum of about 200 molecules per particle. The poliovirus-proflavine complex was photosensitive, the action spectrum corresponding approximately to the absorption spectrum of the free dye. Photoinactivation was oxygen dependent and relatively independent of temperature, pH, and salt concentration. Kinetics of photoinactivation were complex. At higher dye concentrations an initial single-hit, first-order curve was observed, followed by a slower inactivation rate which was also apparently first order. The quantum efficiency of the initial slope was approximately 10 −2 . With low dye concentrations an initial curvilinear semilogarithmic plot, followed by an apparent linear slope, was obtained. The protein coat of the virus was apparently impermeable to the dye since bound proflavine was not removed without disruption of the particles, and mature virus did not bind proflavine nor was it photosensitive in the presence of added proflavine under ordinary conditions.

Base composition of the ribonucleic acids of the three types of poliovirus

Abstract Techniques for HCl hydrolysis and chromatography were modified to permit analysis of the bases in the RNA of representative strains of the three types of poliovirus. The base compositions of these strains were found to be essentially the same, but poliovirus RNA was markedly different from the RNA of host cells.

The separation of infectious and autointerfering particles in vesicular stomatitis virus preparations

Abstract Vesicular stomatitis virus (VSV) infections produce two sizes of particles that differ not only in length, but also in infectivity and autointerference properties. The two forms have been separated by sucrose density gradient centrifugation after partial purification by differential centrifugation, enzyme, and fluorocarbon treatment. Peaks of radioactivity of 32 P-labeled preparations coincided with the two light-scattering bands. Infectivity was associated with the lower band which contained long (L) rods. Autointerference was associated with the upper band which contained short (S) rods.

Calicivirus proteins in infected cells: Evidence for a capsid polypeptide precursor

Abstract Five virus-specific nonstructural proteins were observed in calicivirus-infected cells in addition to the 60,000-dalton capsid polypeptide. They are designated P135, P80, P40, P35, and P29, where the numbers refer to molecular weights in kilodaltons. A new protein, P86, was seen when cells were labeled at 43°. A subsequent 2.5-hr chase at 37° suggested that this protein is a precursor to the capsid polypeptide. Tryptic peptide analysis substantiated this precursor-product relationship. P86 may represent the primary translation product of the 22 S subgenomic mRNA.

Biochemical and biophysical properties of vesicular exanthema of swine virus

Abstract Vesicular exanthema of swine virus (VESV) was purified and some of its biochemical and biophysical properties measured. VESV grew in the presence of 2 μg/ml actinomycin D, was ether resistant, and was not stabilized by cations. Its sedimentation rate was approximately 207 S, and its composition was approximately 20% RNA and 80% protein. The RNA, which was not readily obtained in intact form, appeared to be single stranded with a molecular weight of about 2 × 10 6 . Heterogeneous distributions of VESV were observed upon centrifugation in CsCl density gradients. Crude virus, or virus concentrated by differential centrifugation or by ethanol precipitation, showed a major band at 1.36 g/ml, a secondary band at 1.38 g/ml, and some infectious virus of intermediate density. Sequential treatment with ethanol and chloroform produced a sharp peak at 1.38 g/ml. Chloroform treatment in conjunction with centrifugal concentration produced a sharp band of infectious virus at an intermediate density, 1.37 g/ml, and a band of low infectivity at 1.38 g/ml. The properties of VESV were different from those of the recognized groups of viruses, but most closely resembled properties of the picornaviruses, of which poliovirus and swine enteroviruses were employed for some direct comparisons in these studies.

Assay of antibodies to caliciviruses by radioimmune precipitation using staphylococcal protein A as IgG adsorbent.

SummaryA radioimmune assay method designated St-RIP using a staphylococcal IgG adsorbent, which potentially has broad applications to viral (and nonviral) antigen-antibody systems, was applied to detection of calicivirus antibodies. Purified125I-labeled virions of San Miguel sea lion virus serotypes 4 (SMSV-4) and 5 (SMSV-5) were incubated with sera; the immune complexes were reacted with an immunoadsorbent, formaldehyde-fixed staphylococci (Staphylococcus aureus protein A producer, strain Cowan I), and collected by centrifugation. Broad cross-reactivity was observed among serotypes of SMSV and vesicular exanthema of swine virus (VESV), but there was no reaction with antisera to six noncaliciviruses. Antibody production in a rabbit inoculated with SMSV-5 polypeptide was monitored by St-RIP assay; reactivity with intact SMSV-4 virion antigen was slightly less than, but closely paralleled, reactivity with SMSV-5 virion antigen. Applicability of the St-RIP test to serologic survey was demonstrated with pinniped, swine, and human (laboratory personnel) sera; numerous positive St-RIP reactions suggested the occurrence of widespread contacts with caliciviruses.