Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.

Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles.

In a previous study we demonstrated that vesicular stomatitis virus (VSV) can be used as a vector to express a soluble protein in mammalian cells. Here we have generated VSV recombinants that express four different membrane proteins: the cellular CD4 protein, a CD4-G hybrid protein containing the ectodomain of CD4 and the transmembrane and cytoplasmic tail of the VSV glycoprotein (G), the measles virus hemagglutinin, or the measles virus fusion protein. The proteins were expressed at levels ranging from 23-62% that of VSV G protein and all were transported to the cell surface. In addition we found that all four proteins were incorporated into the membrane envelope of VSV along with the VSV G protein. The levels of incorporation of these proteins varied from 6-31% of that observed for VSV G. These results suggest that many different membrane proteins may be co-incorporated quite efficiently with VSV G protein into budding VSV virus particles and that specific signals are not required for this co-incorporation process. In fact, the CD4-G protein was incorporated with the same efficiency as wild type CD4. Electron microscopy of virions containing CD4 revealed that the CD4 molecules were dispersed throughout the virion envelope among the trimeric viral spike glycoproteins. The recombinant VSV-CD4 virus particles were about 18% longer than wild type virions, reflecting the additional length of the helical nucleocapsid containing the extra gene. Recombinant VSVs carrying foreign antigens on the surface of the virus particle may be useful for viral targeting, membrane protein purification, and for generation of immune responses.

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.

Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Necrotic stomatitis : with special reference to its occurrence in calves (calf diphtheria) and pigs (sore mouth) /

Bibliography: p. 44-48.

Outline for removable partial denture service,

Mode of access: Internet.

Complete denture occlusion: an evidence-based approach

Purpose: This study involved an extensive search for randomized controlled clinical trials comparing bilateral balanced and canine-guided dentures, and questioned whether a bilateral balanced occlusion is imperative for successful denture treatment. Materials and Methods: Studies were identified by searching electronic databases (PubMed/MEDLINE, ISI Web of Science, LILACS, and BBD). The keywords “denture” and “occlusion” were used. The minimum inclusion requirements were (1) randomized controlled trials with patients of any age wearing both maxillary and mandibular conventional complete dentures (CDs), (2) comparison between bilateral balanced and canine-guided dentures, and (3) assessment of masticatory function and/or patients’ satisfaction. Results: The search resulted in the identification of 5166 articles. Subsequently, 5156 articles were excluded on the basis of title and abstract. By the end of the search phase, seven randomized controlled trials were considered eligible. Conclusions: Current scientific evidence suggests that bilateral balanced occlusion is not imperative for successful treatment with conventional CDs in average patients. More studies are necessary to identify if specific clinical conditions may benefit from a balanced occlusion.

The influence of mandibular ridge anatomy on treatment outcome with conventional complete denture.

Visto que indicadores de prognóstico são uma ferramenta importante para a seleção de pacientes a serem tratados com prótese total, este estudo investigou a influência da forma e da resiliência do rebordo alveolar mandibular sobre a retenção e estabilidade de próteses totais convencionais. Noventa e três pacientes desdentados portadores de próteses totais superior e inferior compuseram a amostra. Os dados foram coletados quanto a forma e resiliência do rebordo mandibular. As próteses foram avaliadas para a retenção e estabilidade utilizando-se uma ferramenta objetiva e reproduzível. As associações entre as características clínicas do rebordo alveolar mandibular e retenção e estabilidade das próteses foram analisados por meio dos testes qui-quadrado e exato de Fisher (α = 0.05). Observou-se associação significativa entre a forma do rebordo e a estabilidade da prótese (p <0,05), enquanto que a resiliência foi associada significativamente com a retenção (p <0,001). Baseando-se nos resultados, a resiliência e forma do rebordo mandibular influenciaram, respectivamente, a retenção e estabilidade de próteses totais convencionais.

Longitudinal Clinical Evaluation of Undercut Areas and Rest Seats of Abutment Teeth in Removable Partial Denture Treatment

PURPOSE: Adequate preparation of abutment teeth for removable partial denture (RPD) rest seats allows appropriate masticatory force transmission, retention, and stability of supporting structures. It follows that careful preparation will be important for the longevity of the rehabilitation. The present study aimed to clinically evaluate rest seats and undercut areas of abutment teeth in RPD wearers after 2 years of use. MATERIALS AND METHODS: A total of 193 occlusal, incisal, and cingulum rest seats were evaluated in terms of shape, rest adaptation, wear, caries, fractures, and surface type (enamel, composite resin, or amalgam). Two hundred and fourteen undercut areas were evaluated in terms of surface type (enamel or restoration) and integrity. This study was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte, resolution 196/1996, protocol number 11/05. RESULTS: Intact preparations accounted for 92.2% of the total. Application of the Pearson test (p= 0.289) found no statistically significant differences among the materials on which the rest seats were prepared. For the undercut areas, 20.7% of those obtained on restorative material were nonintact. In addition, Fisher's exact test showed a statistically significant difference (p= 0.001) in surface type; enamel surfaces were shown to be 14 times more stable than restored surfaces. CONCLUSIONS: The results of this study suggest that rest seats are stable, regardless of the material on which they are prepared. Retentive areas were shown to be more stable when they were located in enamel.

Longitudinal Clinical Evaluation of Undercut Areas and Rest Seats of Abutment Teeth in Removable Partial Denture Treatment

PURPOSE: Adequate preparation of abutment teeth for removable partial denture (RPD) rest seats allows appropriate masticatory force transmission, retention, and stability of supporting structures. It follows that careful preparation will be important for the longevity of the rehabilitation. The present study aimed to clinically evaluate rest seats and undercut areas of abutment teeth in RPD wearers after 2 years of use. MATERIALS AND METHODS: A total of 193 occlusal, incisal, and cingulum rest seats were evaluated in terms of shape, rest adaptation, wear, caries, fractures, and surface type (enamel, composite resin, or amalgam). Two hundred and fourteen undercut areas were evaluated in terms of surface type (enamel or restoration) and integrity. This study was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte, resolution 196/1996, protocol number 11/05. RESULTS: Intact preparations accounted for 92.2% of the total. Application of the Pearson test (p= 0.289) found no statistically significant differences among the materials on which the rest seats were prepared. For the undercut areas, 20.7% of those obtained on restorative material were nonintact. In addition, Fisher's exact test showed a statistically significant difference (p= 0.001) in surface type; enamel surfaces were shown to be 14 times more stable than restored surfaces. CONCLUSIONS: The results of this study suggest that rest seats are stable, regardless of the material on which they are prepared. Retentive areas were shown to be more stable when they were located in enamel.

Impact on quality of life of removable partial denture wearers after 2 years of use

The use of clinical indicators of satisfaction (OHIP) can be applied to evaluate the impact of denture use on patient quality of life, since dental problems and disorders interfere in the normal life of individuals. Aim: This study aimed at evaluating the satisfaction level of patients rehabilitated with removable partial dentures (RPD) after 2 years of use. Methods: An observational study was carried out on 28 patients with a mean age of 45 years, treated with RPD at the Department of Dentistry of the Federal University of Rio Grande do Norte in 2005. Patients signed informed consent and answered the Oral Health Impact Profile (OHIP) questionnaire on three occasions: prior to rehabilitation and at 3 months and 2 years of denture use. Repeated-measures ANOVA was applied for data analysis. Results: A difference was found between data obtained at the moment of fitting and three months after denture use (p<0.001). However, no variation was observed when comparing data from 3 months and 2 years of use (p>0.05). The variables of gender and age did not interfere in the result (p>0.05). Conclusions: The degree of patient satisfaction after RPD installation was significant at the moment of fitting and 3 months after denture use, but no significant difference was found between 3 months and 2 years of denture use.

Impact on quality of life of removable partial denture wearers after 2 years of use

The use of clinical indicators of satisfaction (OHIP) can be applied to evaluate the impact of denture use on patient quality of life, since dental problems and disorders interfere in the normal life of individuals. Aim: This study aimed at evaluating the satisfaction level of patients rehabilitated with removable partial dentures (RPD) after 2 years of use. Methods: An observational study was carried out on 28 patients with a mean age of 45 years, treated with RPD at the Department of Dentistry of the Federal University of Rio Grande do Norte in 2005. Patients signed informed consent and answered the Oral Health Impact Profile (OHIP) questionnaire on three occasions: prior to rehabilitation and at 3 months and 2 years of denture use. Repeated-measures ANOVA was applied for data analysis. Results: A difference was found between data obtained at the moment of fitting and three months after denture use (p<0.001). However, no variation was observed when comparing data from 3 months and 2 years of use (p>0.05). The variables of gender and age did not interfere in the result (p>0.05). Conclusions: The degree of patient satisfaction after RPD installation was significant at the moment of fitting and 3 months after denture use, but no significant difference was found between 3 months and 2 years of denture use.