Separation and purification of the mRNAs for vesicular stomatitis virus NS and M proteins.

All five vesicular stomatitis virus mRNAs were separated and purified by acid-urea agarose gel electrophoresis. The identities of the NS and M mRNAs were established by their in vitro translation.     

High multiplicities of infection favor rapid and random evolution of vesicular stomatitis virus

T₁ oligonucleotide mapping of vesicular stomatitis virus RNA shows that the genome undergoes continuous and extensive mutation during repeated high-multiplicity lytic passages in vitro. In contrast, repeated low-multiplicity lytic passages lead to few if any oligonucleotide map changes. Many factors such as intergenomic complementation, selection by defective interfering particles, and genome competition may be involved in the more rapid genome evolution occurring during high-multiplicity passages. In matched high-multiplicity passage series passaged in parallel, the accumulation of genome mutations proceeded not along determined pathways but rather randomly, since dissimilar map changes usually accumulated in matched sets of parallel-passage series. Random virus evolution was also observed in persistently infected cells maintained in parallel cultures, or in vivo as tumors in different nude mice. No attempt is made here to relate genome changes to phenotypes, but numerous interesting phenotypes emerge during persistence. One clone isolated following 70 months of persistent infection replicates very poorly, and produces enormous particle-to-infectious unit ratios. Such phenotypes could be partly responsible for low virus yields, and for difficulties and failures to isolate RNA viruses from persistently infected cells and tissues.     

Promotion of vesicular stomatitis virus nucleocapsid protein production by arthopod saliva

In a previous study (Hajnicka, V. et al., Parasitology 116, 533-538, 1998), the infectivity titer of vesicular stomatitis virus (VSV) was shown to increase up to 10,000-fold when mouse L cells were treated with tick salivary gland extract (SGE) prior to infection. To examine this effect at the level of viral protein production, radiolabeled VSV-infected cells were analyzed by double-dimensional gel electrophoresis. A pre-treatment of cells with SGE from partially fed ticks in amounts corresponding to 1 or 3 salivary glands increased the level of both viral nucleocapsid (N) protein and phosphoprotein (P) in a dose-dependent manner. The effect was more pronounced for N protein and could account for the dramatic increase in infectious virus yield. Promotion of viral infectivity by arthropod saliva may support the arthropode-borne transmission cycle of VSV.     

Pseudotypes of vesicular stomatitis virus and Pichinde virus.

Super-infection of Pichinde virus-infected cells with vesicular stomatitis virus (VSV) resulted in the production of pseudotype virus which was not neutralized by antiserum to VSV but which was neutralized by antiserum to Pichinde virus. Analysis of pseudotype virus production in relation to the kinetics of replication of Pichinde virus demonstrated that pseudotype virus production occurred when super-infection with VSV was initiated 8 h or more after infecting the cells with Pichinde virus. The quantities of pseudotype virus produced correlated with the quantities of Pichinde virus antigen detected on the surface of the cells both during acute infection and in cells chronically infected with Pichinde virus. The observations indicate that pseudotype of VSV and Pichinde virus are readily formed and that the formation of pseudotype virus may be used to examine the Pichinde virus antigens expressed on the surface of infected cells.     

A rapid and inexpensive procedure for the purification of adenovirions

Summary A procedure for the purification of adenovirions is described. About 99 per cent pure adenovirions were obtained within only eight hours by use of chloroform treatment and two cycles of simultaneous sedimentation velocity and isopycnic centrifugation through a glycerol layer into a preformed potassium bromide gradient.With 2 FiguresThis work was supported by the Deutsche Forschungsgemeinschaft.     

Proteins of vesicular stomatitis virus IV. A comparison of tryptic peptides of the vesicular stomatitis group of rhabdoviruses

The methionme-containing tryptic peptides of the five constituent polypeptides of members of the vesicular stomatitis groups of rhabdoviruses were compared to the Indiana serotype by two-dimensional thin-layer chromatography-electrophoresis. While the corresponding protein of each virus serotype could be identified by its characteristic map, there were apparent common peptides shared between serologically related members. Three methionine-containing peptides of the N protein of VSV (Indiana) could not be separated from peptides of VSV (Cocal) and of these three, one peptide was also present in VSV (New Jersey). A comparison of the tryptic peptides of proteins from two members of the VSV (New Jersey) serotype revealed characteristic differences in the polypeptides from the closely related, but serologically distinguishable, New Jersey-Concan and New Jersey Missouri isolates.     

Superinfection exclusion by vesicular stomatitis virus

The infection of baby hamster kidney (BHK21) cells by the Indiana strain of vesicular stomatitis virus (VSV) causes a rapid loss of the ability of the cells to be superinfected by VSV virions or defective-interfering particles. This exclusion phenomenon is at the level of virus penetration and requires viral gene expression and a functional VSV transmembrane glycoprotein G. Infection with the New Jersey serotype of VSV also inhibits the uptake of the Indiana serotype. However, infection of BHK21 cells with either encephalomyocarditis, Newcastle disease, or influenza A viruses does not inhibit superinfection by VSV.     

A slow sedimenting infective component in vesicular stomatitis virus

Summary Evidence has been presented for the presence of a 30 to 40 S infective component in vesicular stomatitis virus preparations. It was found to have a low buoyant density (1.05 g/ml), relatively large size and could multiply in BHK cells to produce complete vesicular stomatitis virus, which suggests that it consists of 625S infective particles (major component) attached to cellular lipids. However, the slow component was not transformed by Tween 80 to infective major component, showing that they probably only differ in their surface characteristics.Submitted by the author in a thesis to Reading University, England, in May 1968, in partial fulfillment of requirements for the degree of Doctor of Philosophy.     

A study of the interference phenomenon in vesicular stomatitis virus replication

Summary Baby hamster kidney cell monolayers were infected with unfractionated vesicular stomatitis virus at input multiplicities ranging from 1 to 1/10⁵. Cells infected at multiplicities of 1/10³ or less produced 10 times as much infective virus but considerably less interfering component than cells infected at multiplicities exceeding 1/100. This has been shown to be due to the presence in the unfractionated virus pool of 2 viruses having different growth rates. The faster growing virus (lag phase 2 hours) does not cause interference, even at multiplicities greater than 100, and does not produce measurable amounts of interfering component in a single passage. The slower growing virus has a lag phase of 3.5 hours and produces large quantities of interfering component in addition to infective virus. This virus will also interfere with viral replication when 10 to 50 particles are added to each cell. A similar number of interfering particles are required to produce the same amount of inhibition. The interfering particle is not self-replicating but is produced only when virus multiplies. The simultaneous presence of infective and interfering particles in a virus harvest is explained by the earlier formation and release of infective virus in the growth cycle.     

Effect of concanavalin A on vesicular stomatitis virus maturation.

Addition of concanavalin A to BHK cell monolayers infected with vesicular stomatitis virus prevented the formation of mature virus particles. In these cells the virus glycoprotein (G) was inserted into the plasma membrane and the protein that is in close association with the ribonucleic acid, protein N, was found in the cytoplasm. At times when cells infected in the absence of the lectin were liberating virus into the supernatant medium, the M or matrix protein was found in association with the plasma membrane of the lectin-treated cells. The removal of the lectin from the cells with alpha-methyl-D-glucoside 3 h after infection was followed by the immediate release of mature virus particles. The rate of virus release from these cells was the same as that from cells infected in the absence of the lectin. Addition of cycloheximide, and inhibitor of protein synthesis, immediately after alpha-methyl-D-glucoside treatment of the cells did not alter the rate of virus production, suggesting that the proteins required for virus synthesis were available in the lectin-treated cells and that virus assembly took place without further protein synthesis on removal of the lectin.     

A vesicular stomatitis pseudovirus expressing the surface glycoproteins of influenza A virus

Pseudotyped viruses bearing the glycoprotein(s) of a donor virus over the nucleocapsid core of a surrogate virus are widely used as safe substitutes for infectious virus in virology studies. Retroviral particles pseudotyped with influenza A virus glycoproteins have been used recently for the study of influenza hemagglutinin and neuraminidase-dependent processes. Here, we report the development of vesicular-stomatitis-virus-based pseudotypes bearing the glycoproteins of influenza A virus. We show that pseudotypes bearing the hemagglutinin and neuraminidase of H5N1 influenza A virus mimic the wild-type virus in neutralization assays and sensitivity to entry inhibitors. We demonstrate the requirement of NA for the infectivity of pseudotypes and show that viruses obtained with different NA proteins are significantly different in their transduction activities. Inhibition studies with oseltamivir carboxylate show that neuraminidase activity is required for pseudovirus production, but not for the infection of target cells with H5N1-VSV pseudovirus. The HA-NA-VSV pseudoviruses have high transduction titers and better stability than the previously reported retroviral pseudotypes and can replace live influenza virus in the development of neutralization assays, screening of potential antivirals, and the study of different HA/NA reassortants.