Pyrophosphate in the 5' terminal position of a viral ribonucleic acid.

A pancreatic ribonuclease digest of carbon-14-labeled Satellite Tobacco Necrosis Virus RNA was fractionated, according to charge, by column chromatography. Individual fractions were dephosphorylated with alkaline phosphomonoesterase and rechromatogramed. The fraction originally containing oligonucleotides with seven negative charges separated into two components corresponding to five and two negative charges, respectively, and therefore must have contained a terminal trinucleotide 5'-pyrophosphate, in addition to the internal hexanucleotides. Other fractions when similarly treated were found to contain only internal oligonucleotides.

Priority setting in Austria.

Priority setting and rationing are not yet matters of public debate in Austria. Before a discussion on rationing is undertaken, the incentive system concerning the financing of health care services needs to be reformed, especially in relation to the reimbursement of hospitals and physicians. This is necessary if a waste of scarce resources is to be avoided. Although methods and principles of priority setting and rationing are not openly discussed, several rationing techniques are already performed as organisational tools. A Eurobarometer survey of the population regarding their satisfaction with the health care system confirms the presumption that Austrians are not aware of the fact that rationing methods already exist.

Cutaneous fibropapillomas of green turtles (Chelonia mydas).

Six juvenile green turtles (Chelonia mydas) from the Indian River Lagoon System, Florida, U.S.A., with multiple cutaneous fibropapillomas, were kept in isolation and examined over a 6-month period. Histologically, the fibropapillomas consisted of a slightly to moderately hyperplastic epidermis overlying a thickened hypercellular dermis. In the earliest lesions, ballooning degeneration was present predominantly in the stratum basale where rete ridges extended into the dermis; aggregates of mixed inflammatory cells were present around dermal vessels. As the lesions matured, they developed an arborizing, papillary pattern. More mature lesions had a less verrucous, often ulcerated surface, with the dermis composed primarily of large collagenous fascicles and relatively few fibroblasts. While numerous trematode eggs were present within dermal capillaries of a histologically similar biopsy specimen from an Hawaiian green turtle, no trematode eggs were observed in any of 28 biopsies examined from the six Florida green turtles in this study. Low stringency Southern blot hybridization and a reverse Southern blot failed to demonstrate papillomavirus DNA in any of the samples extracted. Ultrastructural evaluation of the earliest lesions demonstrated membrane-bound intracytoplasmic vacuoles within epidermal cells in the stratum basale. Similar vacuoles were also observed in the epidermal intercellular spaces and within the dermis. Occasionally, particles with electron-dense centres and measuring 155 to 190 nm were observed in these vacuoles.

Cloning and characterization of a canine oral papillomavirus.

A papillomavirus, isolated from oral papillomas in young Beagles, was used to produce a live-virus vaccine. After the IM use of this vaccine, some dogs developed squamous cell carcinomas at the inoculation site. The virus was isolated from the original vaccine and was cloned into pBR322. A detailed restriction map of the viral genome was generated.

Papillary squamous cell carcinoma in three young dogs.

Papillary squamous cell carcinomas were located on the gingiva of 3 young dogs. The tumors locally invaded the soft tissues of each dog, and invaded bone in 2 dogs. Surgical excision was unsuccessful in eliminating 2 of the tumors. Surgery and radiotherapy were effective, and recurrence has not been observed in 39 months in 1 dog, 32 months in a second, and 10 months in a third. Superficially, the oral masses resembled papillomas, which are known to be caused by viruses. Cytopathologic indication of productive infection was not evident, and papillomavirus antigens could not be detected by immunohistochemical methods. Electron microscopy failed to identify viral particles in 2 of the tumors. High and low molecular weight DNA extracts from 2 of the tumors contained no detectable papillomavirus genome when probed under conditions of either high or low stringency by Southern blot hybridization with a cloned canine oral papillomavirus genome.

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells.

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.

UV inactivation of the biological activity of defective interfering particles generated by vesicular stomatitis virus.

UV inactivation of vesicular stomatitis virus and its defective interfering (DI) particles was measured in order to obtain the target size for interference. In the case of DI particles whose genomes mapped at the 5' end of the virion RNA, this target size corresponded to the entire DI particle RNA molecule regardless of whether it amounted to 10, 30, or 50% of the viral genome. These data were interpreted as demonstrating that both termini of the DI particle RNAs were required for their replication and for interference with virion RNA replication. The unique heat-resistant DI particle, with an RNA molecule corresponding to the 3' half of the viral genome, exhibited an inactivation target size of approximately 42% of its RNA molecule with respect to both homotypic and heterotypic interference. Unlike other DI particles, this particle interfered with virion primary transcription. The unusual inactivation target size of the heat-resistant DI particle was interpreted as being a compromise between the requirements for replication of its genome and those for interference with virion primary transcription.

In vitro and in vivo inhibition of primary transcription of vesicular stomatitis virus by a defective interfering particle.

A unique defective interfering (DI) particle, generated by a heat-resistant (HR) mutant of Indiana serotype vesicular stomatitis virus, was capable of inhibiting primary transcription by heterologous New Jersey serotype virions. The correlation between this phenomenon and the lowering of viral yields from doubly infected cells was investigated by the construction of chimeric DI particles containing the HR DI particle genome with a thermolabile polymerase. At the nonpermissive temperature, these DI particles were unable to self-transcribe, inhibit virion primary transcription, or reduce virion yield, but were able to be replicated. These results suggested that self-transcription of the HR DI particle genome was a prerequisite for heterotypic interference, but not for its own replication. Inhibition of virion primary transcription by HR DI ribonucleocapsids was also observed in vitro. At low HR DI to virion ribonucleocapsid ratios, the extent of inhibition was concentration dependent, whereas at high ratios, the amount of inhibition was concentration independent, approaching a limiting maximum value. A speculative mathematical model, which quantitatively accounts for these data, is presented. According to this model, the higher affinity for polymerase molecules by the HR DI ribonucleocapsids is explained in terms of dissociation events during transcription, which are more frequent in the longer virion ribonucleocapsids.

Characterization of vesicular stomatitis virus mutants by partial proteolysis.

Structural proteins of temperature-sensitive (ts) mutants of vesicular stomatitis virus, Indiana serotype, were compared with those of wild-type and revertant virions by electrophoresis on polyacrylamide gels of partial digests with Staphylococcus aureus V8 protease. Mutants of complementation groups III (tsG31 and tsG33), II (tsG22), and IV (tsG41) differed from the wild-type virion in peptide profiles of their M, NS, and N proteins, respectively. The differences were only detectable over a narrow range of enzyme-substrate ratios and were due to peptides transiently generated during incomplete digestion. Proteins of revertants to tsG31, tsG22, and tsG41 exhibited the wild-type virion peptide pattern, indicating that reversion had restored their original conformation. However, in the case of tsG22, the NS peptide profile reverted to the wild-type phenotype only partially, suggesting that a silent mutation might have taken place during either the original chemical mutagenesis or the following repeated laboratory passages. The apparent alteration in protein conformation and its restoration upon reversion of the mutants indicated that the lesions of groups III and IV were located in the M and N proteins, respectively. Moreover, for the first time, the site of mutation of group II could be positively identified as the NS protein cistron.

Identity of the 5'-terminal RNA nucleotide sequence of the satellite tobacco necrosis virus and its helper virus: possible role of the 5'-terminus in the recognition by virus-specific RNA replicase.

A pancreatic ribonuclease digest of (14)C-labeled tobacco necrosis virus RNA was fractionated according to charge by column chromatography. Individual fractions were dephosphorylated with alkaline phosphatase and rechromatographed. The fraction, originally containing oligonucleotides with seven negative charges, separated into two components corresponding to five (-5) and two negative charges (-2). The -5 fraction was derived from the internal oligonucleotides while the -2 fraction must have originated from a 5'-pyrophosphorylated terminal trinucleotide. The sequence of this terminal trinucleotide was determined by column chromatography on DEAE-cellulose in a triethyl ammonium carbonate gradient, using the appropriate markers. The radioactivity chromatographed with a (ApGp)U marker. The order of the Ap and Gp was determined after ribonuclease T(1) and alkaline phosphatase digestion. The radioactivity in the product chromatographed with an ApG marker. The 5'-terminus of tobacco necrosis virus RNA was therefore determined as ppApGpUp..., which is identical to the terminus of the RNA of its satellite virus as previously determined (J. Mol. Biol., 38, 59 (1968); Science, 160, 1452 (1968)). The 5' pyrophosphate in both viruses was probably formed by an in vivo enzymatic removal of a gamma-phosphate from a triphosphate, and its presence in both viruses suggested a common site of synthesis. The identity of the 5'-terminal sequences is considered not to be fortuitous and is discussed from the standpoint of their role as a recognition site for the virus-specific RNA replicase.

RNA synthesis in temperature-sensitive mutants of vesicular stomatitis virus.

T-particle-free stocks of temperature-sensitive mutants representing the four Glasgow complementation groups of the Indiana serotype of vesicular stomatitis virus were used to study RNA synthesis at the permissive and nonpermissive temperatures of 31 and 39 C, respectively. Mutants selected from the four Glasgow complementation groups were characterized on the basis of particle and ribonucleoprotein formation. Intracellular RNAs were further characterized by polyacrylamide gel electrophoresis. ts G22 (group II) and ts G41 (group IV), previously characterized as RNA negative at the nonpermissive temperature, synthesized low levels of RNA which could not be attributed to contaminating levels of revertants. Furthermore, the levels of synthesis could not be reduced by the addition of cycloheximide. These data suggest that ts G22 (group II) and ts G41 (group IV) contain a thermally stable, virion-encapsidated transcriptase, but fail to amplify RNA synthesis due to a thermally labile function presumably necessary for the synthesis of viral RNA. ts G31, a group III mutant, synthesized intracellular RNA at amplified levels at the nonpermissive temperature. Intracellular ribonucleoprotein complexes were isolated in copious amounts; however, no particles corresponding in size to finished virions were observed. These data suggest a thermally labile maturation factor or envelope associated structural protein to be defective in ts G31 (group III). ts G11 (group 1) showed no detectable RNA synthesis at the nonpermissive temperature. These data suggest ts G11 (group I) contains a thermally labile component involved in early transcription. This group may contain a number of mutants defective in different components of the transcription apparatus, which may not complement in vivo because of the physical improbability of subunit exchange between virion particles of the incoming inoculum.

Characterization of New Jersey vesicular stomatitis virus isolates from horses and black flies during the 1982 outbreak in Colorado.

Vesicular stomatitis viruses isolated from horses, afflicted during the recent outbreak in the western United States, and from black flies (Simuliidae) were characterized with respect to the homology of their genomic RNAs and the mobility of their proteins in polyacrylamide gels. All the isolates were very similar, if not identical, with respect to these two parameters. When the black fly isolate was compared to other VSV isolates, this virus appeared to belong in the Hazelhurst subgroup of the New Jersey serotype of VSV. Since the other viruses in this division were obtained from infected swine, the natural host range of this subgroup has been extended to horses.

The impact of the Austrian hospital financing reform on hospital productivity: empirical evidence on efficiency and technology changes using a non-parametric input-based Malmquist approach.

The 1997 hospital financing reform has been supposed to reduce considerable inefficiencies in the provision of hospital care in Austria. This paper focuses on the changes in hospital productivity between 1994 and 1998, thus including three years before the reform and two years after the reform. Using Data Envelopment Analysis we calculated the input-based Malmquist index, which is then decomposed into indices of pure technical efficiency change, scale efficiency change and technology change. The results illustrated a considerably positive shift in technology between 1996 and 1998, whereas the intended enhancement in technical efficiency has not yet taken place.