Effects of hydrostatic pressure on a membrane-enveloped virus: high immunogenicity of the pressure-inactivated virus.

A new approach to the preparation of antiviral vaccines relying on the inactivation of the virus particle by hydrostatic pressure is described. The enveloped virus vesicular stomatitis virus was utilized as a model; a pressure of 260 MPa applied for 12 h reduced infectivity by a factor of 10(4), and the antibodies against pressurized material were as effective as those against the intact virus when measured by their neutralization titer. Fluorescence measurements indicate that application of pressure results in perturbations of the particle interactions that permit binding of specific molecular probes. Electron microscopy showed that the membrane of the pressurized virus was partially preserved, presenting the spike pattern of the membrane G protein. Unlike the icosahedral viruses, dissociation into smaller particles was not observed, but a constant change in the morphology was the presence of a bulge in the surface of the pressurized virus, indicating a displacement of the capsid subunits, retained under the lipid and protein membrane.     

Effects of humic materials on virus recovery from water.

Humic and fulvic acids were tested for their ability to interfere with virus recovery by microporous filters. Two electropositively charged types of filter (Seitz S and Zeta Plus 60S) were used to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in high-volume experiments, humic acid led to premature blockage of the filters. The efficiency of virus recovery by a second concentration step, organic flocculation of the filter eluate, was tested. For all the filter types tested, this procedure was not affected by the presence of humic or fulvic acid in the input water.     

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.     

Effects of temperature and antibody on the cyclid growth of vesicular stomatitis virus.

To see the effects of temperature on the interrelated cyclic production of standard and defective interfering (DI) particles of vesicular stomatitis virus, a temperature-sensitive (ts) G114 mutant was passaged successively at different temperatures and the production of the two types of viral particles as well as the ability of Chinese hamster ovary cells to survive each passage was continuously monitored. When the temperature was nonpermissive for standard virus, the synthesis of both standard and defective interfering particles was inhibited. When revertants appeared in the population, their ability to take over the infection depended on the permissiveness of the temperature for the temperature-sensitive mutant. At permissive temperatures periodic inhibition of both types of standard viruses was maintained by the production of defective interfering particles. Reverents did not become a majority of the population due to this periodic inhibition. When the conditions were nonpermissive for the mutant, revertants became the major standard virus in the population within a few passages. These findings can be understood if conditions of high and low multiplicities are dissected out together with a thorough understanding of the individual properties of each of the viral particles and of the result of interactions between them. In the presence of antiserum which neutralized only 90% of the viral particles, cyclic production of standard virus occurred, with a decline in the total amount of virus produced after each cycle. Therefore, in the presence of limiting concentrations of antiserum, the virus appeared to be able to establish a persistent cyclic growth pattern.     

Effects of organic matter on virus transport in unsaturated flow.

The effects of natural humic material and sewage sludge organic matter (SSOM) derived from primary treated sewage sludge on virus transport by unsaturated flow through soil columns were evaluated. Bacteriophage MS-2 was applied to loamy fine sand columns 0.052 m in diameter and 1.05 m long. Virus concentrations in the influent and effluent were measured daily for 7 to 9 days. In the first experiment, virus transport through two fresh soil columns was compared with that through a column previously leached with more than four pore volumes (T) of well water. The soil water organic matter concentrations in the leachate of the fresh soil declined with time. Relative virus concentrations (C/Co) from one fresh soil column reached 0.82 in 0.9 T and then declined to 0.51 by 2.1 T. The other fresh soil column reached and maintained a steady-state relative virus concentration [(C/Co)s] of 0.47 from 1.5 to 2.5 T. The leached column reached and maintained a (C/Co)s of 0.05. Concentrations measured at 0.2-, 0.4-, 0.8-, and 1.05-m depths indicated that most virus particles were removed in the surface 0.2 m. In the second experiment, one leached column was pretreated with SSOM derived from primary treated sewage sludge and the other leached column was untreated. SSOM concentrations declined with depth. A suspension of virus and SSOM in well water was applied to both columns. Although the (C/Co)s values were similar (0.41 for the pretreated column and 0.47 for the untreated column), breakthrough was delayed for the untreated column. Both natural humic material and sewage sludge-derived SSOM increased the unsaturated-flow transport of MS-2.     

Effects of pure interferons on Epstein-Barr virus infection in vitro.

Pure human gamma-interferon as well as alpha-interferon inhibited induction of immunoglobulin synthesis by Epstein-Barr virus but not by pokeweed mitogen in B lymphocytes from adult but not from newborn humans. The interferons inhibited the infected B lymphocytes directly, irrespective of the Epstein-Barr virus immune status of the donor, and their inhibitory effect was synergistic.     

Effects of purified measles virus components on proliferating human lymphocytes.

Preparations of purified measles virus and both core and membrane-rich fractions suppressed mitogen-induced proliferation of human lymphocytes without reducing viability or the number of T lymphocytes in culture. The suppressive activity of the measles virus was heat labile. The virus preparations by themselves did not induce lymphocyte proliferation.     

Effects of organic matter on virus transport in unsaturated flow.

The effects of natural humic material and sewage sludge organic matter (SSOM) derived from primary treated sewage sludge on virus transport by unsaturated flow through soil columns were evaluated. Bacteriophage MS-2 was applied to loamy fine sand columns 0.052 m in diameter and 1.05 m long. Virus concentrations in the influent and effluent were measured daily for 7 to 9 days. In the first experiment, virus transport through two fresh soil columns was compared with that through a column previously leached with more than four pore volumes (T) of well water. The soil water organic matter concentrations in the leachate of the fresh soil declined with time. Relative virus concentrations (C/Co) from one fresh soil column reached 0.82 in 0.9 T and then declined to 0.51 by 2.1 T. The other fresh soil column reached and maintained a steady-state relative virus concentration [(C/Co)s] of 0.47 from 1.5 to 2.5 T. The leached column reached and maintained a (C/Co)s of 0.05. Concentrations measured at 0.2-, 0.4-, 0.8-, and 1.05-m depths indicated that most virus particles were removed in the surface 0.2 m. In the second experiment, one leached column was pretreated with SSOM derived from primary treated sewage sludge and the other leached column was untreated. SSOM concentrations declined with depth. A suspension of virus and SSOM in well water was applied to both columns. Although the (C/Co)s values were similar (0.41 for the pretreated column and 0.47 for the untreated column), breakthrough was delayed for the untreated column. Both natural humic material and sewage sludge-derived SSOM increased the unsaturated-flow transport of MS-2.     

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.     

Effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin.

Mutagenesis studies indicated that the three cytoplasmic cysteines of the influenza virus A/Japan/305/57 hemagglutinin (HA) are all palmitylated, but to an unequal extent. Replacement of all three cysteines abolished palmitylation, but affected neither HA biosynthesis nor function. Palmitate was not required for HA to be incorporated into virions.     

Effects of the 'fusion peptide' from measles virus on the structure of N-methyl dioleoylphosphatidylethanolamine membranes and their fusion with Sendai virus.

31P nuclear magnetic resonance spectroscopy (31P-NMR) was used to study phospholipid organization in hydrated preparations of N-methyl dioleoylphosphatidylethanolamine and a 'fusion peptide' with the sequence: FAGV-VLAGAALGVAAAAQI, which corresponds to the amino terminus of the F1 subunit of the membrane fusion protein of measles virus. These amino acids are believed to mediate syncytia formation, host-cell penetration and hemolysis by infectious virus. The presence of the peptide at 0.5 mole percent significantly facilitated the formation of isotropic 31P resonances. The effects at 1 mole percent peptide were substantially enhanced over the effects observed at 0.5 mole percent, leading to a decrease in the onset temperature of the formation of the isotropic 31P-NMR resonances by about 30 degrees C. The formation of such isotropic 31P-NMR resonances has been previously associated with an increased rate of fusion of large unilamellar vesicles composed of N-methyl dioleoylphosphatidylethanolamine. Enhanced fusion of octadecyl rhodamine-labelled Sendai virus with N-methyl dioleoylphosphatidylethanolamine large unilamellar vesicles was observed when the 'fusion peptide' was incorporated into the large unilamellar vesicles.     

Effects of the open reading frames in the Rous sarcoma virus leader RNA on translation.

Three short open reading frames (ORFs) reside in the 5' leader of Rous sarcoma virus (RSV) and are conserved in all avian sarcoma-leukosis retroviruses. Both extensions of the lengths of the ORFs and alterations in their initiation codons affect viral replication and gene expression. To determine whether the effects on viral replication were due to translational regulation mediated by the ORFs, we examined translation following mutation of the initiation and termination codons of each of the three ORFs. We found that the ORFs marginally enhanced downstream gene expression. Moreover, repression of downstream gene translation was proportional to the lengths of the elongated ORFs and depended on the initiation contexts of the AUG codons. Although the ORFs play a major role in viral activities, their effects on translation were relatively minor. Rather, the ORFs may affect the fate of unspliced avian retroviral RNA in chronically infected cells by participating in the sorting of viral RNA for either translation or encapsidation into virions.     

Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines.

Increased levels of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6, have been detected in specimens from human immunodeficiency virus type 1 (HIV-1)-infected individuals. Here we demonstrate that HIV-1 activates the expression of TNF but not of IL-1 and IL-6 in acutely and chronically infected T cells. The increase in TNF gene expression is due to activation of the TNF promoter by the viral gene product Tat. Transactivation of TNF gene expression requires the product of the first exon of the tat gene and is cell type independent. T cells chronically infected with pol-defective HIV-1 provirus constitutively express both Tat and TNF at levels significantly higher (fivefold) than those seen in control cells, and treatment with phorbol myristate acetate greatly enhances Tat expression and TNF production. As TNF can increase the production of IL-1 and IL-6 and these inflammatory cytokines all enhance HIV-1 gene expression and affect the immune, vascular, and central nervous systems, the activation of TNF by Tat may be part of a complex pathway in which HIV-1 uses viral products and host factors to increase its own expression and infectivity and to induce disease.     

Effects of monensin on morphogenesis and infectivity of Friend murine leukemia virus.

The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious virus was inhibited by treatment of Friend murine leukemia virus-infected Eveline cells with the sodium ionophore monensin. Virus yields were reduced more than 50-fold by 10(-5) M monensin, whereas particle production was reduced by 50% in monensin-treated cells. The resulting particles failed to incorporate newly synthesized gp70 and p15(E), whereas the other structural proteins, p30, p15, p12, and p10, were incorporated into virions. However, monensin did not inhibit the incorporation into virions of preformed gp70. A reduction in the efficiency of cleavage of the PrENV glycoprotein precursor and a defect in the processing of simple endo-H-sensitive to complex endo-H-resistant oligosaccharides suggest that intracellular transport of gp70 may be blocked before its entry into the Golgi apparatus. Fewer particles were found to bud from the cell surface, but intracellular vacuoles with budding virions were detected. Ferritin labeling and pulse-chase studies suggested a cell surface origin for these vacuoles. These experiments indicate that monensin inhibits the transport of Friend murine leukemia virus glycoproteins at an early stage, with a resultant block in the assembly and release of infectious virus.     

Effects of hydrostatic pressure on the structure and biological activity of infectious bursal disease virus.

The effects of high hydrostatic pressure on the structure and biological activity of infectious bursal disease virus (IBDV), a commercially important pathogen of chickens, were investigated. IBDV was completely dissociated into subunits at a pressure of 240 MPa and 0 degrees C revealed by the change in intrinsic fluorescence spectrum and light scattering. The dissociation of IBDV showed abnormal concentration dependence as observed for some other viruses. Electron microscopy study showed that morphology of IBDV had an obvious change after pressure treatment at 0 degrees C. It was found that elevating pressure destroyed the infectivity of IBDV, and a completely pressure-inactivated IBDV could be obtained under proper conditions. The pressure-inactivated IBDV retained the original immunogenic properties and could elicit high titers of virus neutralizing antibodies. These results indicate that hydrostatic pressure provides a potential physical means to prepare antiviral vaccine.