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PubMed1995

Requirements for optimal expression of secreted and nonsecreted recombinant proteins in vaccinia virus systems.

Selection of an optimal promoter is necessary for efficient expression of foreign genes with vaccinia virus. Since a variety of powerful (homologous) vaccinia virus promoters and foreign (heterologous) promoter systems have been described for use in vaccinia, we have addressed the question of whether a general rule exists that allows the prediction of the optimal promoter/gene combination. We have compared the expression properties of four secreted proteins, the human blood clotting factor IX (FIX), the human blood glycoprotein Protein S (ProtS), the human von Willebrand factor (vWF), and the Hepatitis B virus (HBV) middle surface glycoprotein preS2, with proteins that were reported not to be secreted, the HBV large surface glycoprotein preS1 and the murine leukemia virus (MuLV) BM-5 Eco gag protein. In addition, we have included in our study an internal control protein, the vaccinia virus p11 protein, to monitor possible side effects of the promoter system used. Genes encoding the foreign proteins were placed either under control of a synthetic vaccinia virus early/late promoter (selP) or under control of the bacteriophage T7 promoter (T7/emc system). The secreted proteins were more efficiently expressed when fused to the homologous promoter. Direct comparison of the two promoters indicated that the expression level ranged between 1.4 (ProtS) and 3.9 (FIX)-fold higher with the selP than with the T7 promoter. In contrast, the cell-associated HBV preS1 was more efficiently expressed under the T7 promoter and the MuLV BM-5 Eco gag polypeptide was expressed equally well from both promoters. These data indicate that a careful prediction of optimal promoter/foreign gene combinations for the vaccinia virus expression system is possible. The choice of the optimal promoter/expression system is based on a simple classification scheme, discriminating secreted and nonsecreted proteins.

Animals↗
PubMed2005

Choosing appropriate substitution models for the phylogenetic analysis of protein-coding sequences.

Although phylogenetic inference of protein-coding sequences continues to dominate the literature, few analyses incorporate evolutionary models that consider the genetic code. This problem is exacerbated by the exclusion of codon-based models from commonly employed model selection techniques, presumably due to the computational cost associated with codon models. We investigated an efficient alternative to standard nucleotide substitution models, in which codon position (CP) is incorporated into the model. We determined the most appropriate model for alignments of 177 RNA virus genes and 106 yeast genes, using 11 substitution models including one codon model and four CP models. The majority of analyzed gene alignments are best described by CP substitution models, rather than by standard nucleotide models, and without the computational cost of full codon models. These results have significant implications for phylogenetic inference of coding sequences as they make it clear that substitution models incorporating CPs not only are a computationally realistic alternative to standard models but may also frequently be statistically superior.

Classification↗
PubMed1987

4-Ipomeanol-induced effects on Sendai viral pneumonia in mice.

The effects of the pulmonary toxicant, 4-ipomeanol (4-IP), on Sendai viral pneumonia was examined in young adult female C57BL/6J mice. The histologic severity of the pneumonia was closely correlated with increasing doses of the compound. The more severe pneumonia observed in the 4-IP-treated animals was associated with higher pulmonary viral titers and a more diffuse distribution of viral antigen in bronchioles and alveolar parenchyma. There was no difference in the systemic humoral immune response against Sendai virus or pulmonary interferon production in animals that had received 4-IP or vehicle pretreatment. Classification of the antigen-positive cell types in the alveolar parenchyma resulted in a marked relative increase in the numbers of antigen-positive macrophages, as compared with other antigen-positive cell types.

Animals↗
PubMed2002

Polycistronic viral vectors.

Traditionally, vectors for gene transfer/therapy experiments were mono- or bicistronic. In the latter case, vectors express the gene of interest coupled with a marker gene. An increasing demand for more complex polycistronic vectors has arisen in recent years to obtain complex gene transfer/therapy effects. In particular, this demand is stimulated by the hope of a more powerful effect from combined gene therapy than from single gene therapy in a process whose parallels lie in the multi-drug combined therapies for cancer or AIDS. In the 1980's we had only splicing signals and internal promoters to construct such vectors: now a new set of biotechnological tools enables us to design new and more reliable bicistronic and polycistronic vectors. This article focuses on the description and comparison of the strategies for co-expression of two genes in bicistronic vectors, from the oldest to the more recently described: internal promoters, splicing, reinitiation, IRES, self-processing peptides (e.g. foot-and-mouth disease virus 2A), proteolytic cleavable sites (e.g. fusagen) and fusion of genes. I propose a classification of these strategies based upon either the use of multiple transcripts (with transcriptional mechanisms), or single transcripts (using translational/post-translational mechanisms). I also examine the different attempts to utilize these strategies in the construction of polycistronic vectors and the main problems encountered. Several potential uses of these polycistronic vectors, both in basic research and in therapy-focused applications, are discussed. The importance of the study of viral gene expression strategies and the need to transfer this knowledge to vector design is highlighted.

Artificial Gene Fusion↗
PubMed2001

Classification of cell lineage and anatomical site, and prognosis of extranodal T-cell lymphoma -- natural killer cell, cytotoxic T lymphocyte, and non-NK/CTL types.

Due to their minority among the non-Hodgkin lymphomas, classification of extranodal T-cell lymphomas, including those of the natural killer (NK) cell type, has long been controversial and unclear, and the clinical outcome is not well clarified. Recently, new well-defined disease entities have been described based on tumor cell biology combined with anatomical site, clinical features, Epstein-Barr virus (EBV) status, and cell lineage as determined by immunophenotype and genotype. Cytological features are usually not specific, and there are no morphologic correlates with the classification of extranodal T/NK-cell lymphomas. From a human T-cell lymphotropic virus type 1 (HTLV-1) endemic area in Japan, we report here the analysis of 144 cases of extranodal T-cell lymphoma, from which fresh tissues were available. As the clinicopathological features were known, we simply reclassified the cases according to cell lineage and anatomical site. The extranodal T-cell lymphomas were classified into three types on the basis of cell lineage: (1) natural killer cell (NK) type [sCD3-, CD56+, T-cell receptor gene (TCR) germline], (2) cytotoxic T lymphocyte (CTL) type [sCD3+, TIA-1+, TCR rearranged, CD8+/-, CD4-/+], and (3) non-NK/CTL type [sCD3+, TIA-1-, TCR rearranged, CD4+/-, CD8-/+]. In addition to cell lineage, the anatomical site and clinical features were added for subclassification. NK type tumors (35 cases) included the lymphoblastic type, nasal/nasal-type NK lymphoma, and NK leukemia. The CTL type (46 cases) included anaplastic large cell lymphoma (ALCL), cutaneous type, intestinal, gamma delta T-cell type, and an unspecified type. The non-NK/CTL type (63 cases) included adult T-cell leukemia/lymphoma (ATLL), mycosis fungoides (MF), and an unspecified type. With the exception of ATLL and MF, most extranodal T-cell lymphomas had a cytotoxic phenotype of NK type or CTL type and were often associated with EBV infection. MF and the unspecified type within the non-NK/CTL tumors, with the exception of ATLL, had a favorable prognosis. However, NK and CTL types, with the exception of ALCL, were associated with a poor prognosis. Our results indicate that anatomical site and cell lineage are useful predictors of clinical outcomes of extranodal T-cell lymphomas.

Antigens, CD↗
PubMed2001

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada.

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Animals↗
PubMed2001

Pre- and in-hospital management of community-acquired pneumonia in southern France, 1998-99.

A prospective, hospital-based, multicenter study was undertaken to identify the reasons for hospital admission, to describe antibiotic treatment before and during hospitalization, and to determine the outcome of community-acquired pneumonia (CAP). Data collected included prehospital management of CAP, Pneumonia Outcome Research Team (PORT) classification on admission, in-hospital antibiotic treatment, and predictors of death within 30 days. Among the 215 patients (mean age, 66.7 years; M:F ratio, 1.1) recruited, 24 (11.2%) were living in nursing homes. CAP had been diagnosed prior to admission in 55 (25.6%) patients. At admission, 75 (34.9%) patients had a low risk of death (PORT classification I-II). A pathogen was isolated for 55 (25.6%) patients, primarily Streptococcus pneumoniae (n=18), atypical agents (n=16), influenza virus (n=10), and respiratory syncytial virus (n=4). Amoxicillin (with or without clavulanate), cefotaxime, or ceftriaxone monotherapy was prescribed to 121 (56.3%) patients. Dual combination therapy was prescribed to patients at higher risk of death (PORT classification III-V; OR, 3.09). Mortality was 7%. Logistic-regression analysis identified nursing-home residency (OR, 8.36), serum creatinine > or =88 micromol/l (OR, 7.88), and Pneumonia Outcome Research Team classification (OR, 1.02) as independent predictors of death. CAP remains a serious disease for elderly persons living in nursing homes. This population should benefit from immunization with pneumococcal and influenza vaccines.

Adolescent↗
PubMed2005

[Molecular biological identification of Batai virus isolated in China].

BACKGROUND: To study the molecular characteristics of YN92-4 strain isolated from mosquitoes in Yunnan Province and define its classification. METHODS: The S segment of YN92-4 strain was amplified and sequenced by 2 different sets of primers. The phylogenic tree of S fragment was constructed by Phylip bio-software. The amino acid sequences of N and NSs proteins were also studied. RESULTS: YN92-4 strain could be amplified by 2 sets of primers respectively, S segment showed a highest homology with Batai virus (X73464), reached 96.4%, the homology of protein N and NSs amio-acid sequence with Batai virus was 99.1% and 98% respectively. CONCLUSION: The YN92-4 strain belongs to Batai virus, this is the first report of molecular biological identification of Batai virus in China.

Amino Acid Sequence↗
PubMed1988

Use of polyclonal and monoclonal antibodies and analysis of viral RNA in the detection of unusual group A human rotaviruses.

Immuno-enzymatic assay employing monoclonal antibodies and viral RNA analysis by gel electrophoresis were used to classify human group A rotaviruses (HRV) into subgroups I and II. Of 249 fecal samples positive for group A rotaviruses, 29 (11%) belonged to subgroup I and 215 (85%) were identified as subgroup II. Two samples (Ro-302 and Ro-500) contained mixed infections of the two subgroups. Three isolates belonged to neither one of the two subgroups, but they did not yield enough viral RNA to allow their classification. One subgroup I isolate (Ro-1845) contained components typical of subgroup II viruses in that it was identical to serotype 3 and yielded RNA with fast-moving 10th and 11th segments. After growth in culture, the two mixed infections yielded subgroup II viruses, which were identified as serotype 1. The three unclassified isolates grew poorly in culture and could not be further analyzed. The subgroup I isolate (Ro-1845) grew well in culture and yielded virus similar to the original one.

Antibodies↗
PubMed1996

Viral isolates derived from simian varicella epizootics are genetically related but are distinct from other primate herpesviruses.

Epizootics of a natural varicella-like disease occur in populations of nonhuman primates. Several primate herpesviruses have been isolated from these epizootics, but the relatedness of these isolates to each other is not well-defined. In this study, we demonstrated that the restriction endonuclease (REn) profiles of four epidemiologically distinct isolates were similar, although not identical, indicating that simian varicella epizootics are caused by various strains of simian varicella virus (SVV). The genetic variation among the isolates did not map to a specific region of the SVV genome and REn differences were detected within the SVV DNA long component and the inverted repeat region. Southern blot hybridization demonstrated that SVV is more closely related to varicella-zoster virus than to other primate herpesviruses. The study indicates that the current herpesvirus classification scheme should be changed to include SVV as a single taxonomic group within the Varicellovirus genus of alphaherpesviruses. In addition, REn profiles of SVV isolates, derived from primary and secondary episodes of simian varicella in the same monkey, were identical, providing evidence for SVV reactivation in a latently infected monkey.

Animals↗
PubMed2001

Task profile and risk of occupational hepatitis A infection in sewerage workers.

OBJECTIVE: The aim of the study was to assess to what extent parameters of task-related occupational exposure influence anti-hepatitis A virus (anti-HAV) seroprevalence in sewerage workers, using a new instrument for classification of exposure. METHODS: A new instrument for the assessment of work-related infection hazards was developed based on expert interviews, evaluation of literature and theoretical considerations. It was included in a questionnaire for collecting detailed information on occupational exposure, safety awareness, safety behaviour and socio-demography. Anti-HAV status was assessed for all (n = 343) (non-vaccinated) study participants. RESULTS: Marked differences in task profile and task-related exposure within the group of sewerage workers were found, underlining the necessity of a detailed exposure analysis. In a multivariate model three risk factors that were related significantly to anti-HAV positivity were identified: age, country of origin and task-related exposure. CONCLUSIONS: Since task profiles and occupational exposure differ strongly within the job category of sewerage workers. evaluation of endangerment has to reflect individual task-related exposure. The task-exposure matrix developed and presented in this study is a practicable and valid instrument for exposure assessment and may be used for the exposure analysis of further biological agents in this working environment. Besides the known risk parameters age and origin, our study demonstrates a dose-response relationship between the degree of occupational exposure and the anti-HAV seroprevalence. Therefore, an effective worksite HAV-prevention programme should consider all technical, structural and educational measures that help to reduce individual exposure.

Adult↗
PubMed2003

Cloning and sequencing of Duck circovirus (DuCV).

The genome of Duck circovirus (DuCV) is circular and 1996 nts in size. Two major open reading frames were identified, encoding the replicase (V1) and the capsid protein (C1). A stem-loop structure comprising the nonamer 5'-TATTATTAC, conserved in all circo-, nano- and geminiviruses, was found. Unique to DuCV, the region between the 3'-ends of the rep and cap gene contains four repeats of a 44-bp sequence. Phylogenetic analysis shows close relation of DuCV with Goose circovirus and suggests classification of DuCV as a new member of the genus Circovirus of the virus family Circoviridae.

Amino Acid Sequence↗
PubMed2004

Liver transplant recipients mortality on the waiting list: long-term comparison to Child-Pugh classification and MELD.

We sought to evaluate our experience concerning the high waiting list mortality rate for orthotopic liver transplantation (OLT) using the MELD (Model for End-Stage Liver Disease), which has been shown to predict short-term survival better than Child-Turcotte-Pugh (CTP) classification. The predominant end-stage disease was cirrhosis due to hepatitis C virus (67%), patient mean age was 36.8 years, and 72.1% were men. When the patients were included on a waiting list, the MELD score was stratified into W: 0 to 10; X: 11 to 20, and Y: 21 to 40 and the CPT as A: 5 to 6, B: 7 to 9, and C: 10 to 15. It was also observed that 77.8% of patients were on the waiting list, 16.4% underwent OLT and 5.8% had been removed. The estimated survival rate after 1 year was W = 85.4%; X = 83.3%, Y = 46.8%; A = 81.3%, B = 84.2%, C = 45.9%. Child median score was 8 +/- 1.5 (5 to 15) and the MELD was 14.7 +/- 5.1 (8 to 43). The mortality rate was 20.2%. Severe patients classified as Y or C showed greater mortality than the other groups (P <.001), but no significant difference between Y and C strata. The mortality rate was the same as in previous years.

Adolescent↗
PubMedDate not supplied

Psychiatric care for patients with HIV infection. The varying perspectives.

This article reviews the literature on the classification and treatment of psychiatric morbidity associated with infection from the human immunodeficiency virus (HIV). The psychiatric disorders seen in HIV-infected patients are formulated by using one of the following four perspectives as treatment guides: 1) the syndromal or disease perspective, 2) the dispositional or personality perspective, 3) the behavioral or addictive perspective, and 4) the life story perspective.

Antipsychotic Agents↗
PubMed1999

The implications of antigenic diversity for vaccine development.

The reactivity of human monoclonal and polyclonal anti-HIV-1 antibodies demonstrates that shared epitopes, including those that induce neutralizing antibodies, exist and are recognized by the human immune system. A priori, there is no reason why cross-clade neutralizing antibodies could not be induced by an appropriately constructed HIV vaccine. But to construct such a vaccine, it is critical to understand, as completely as possible, the antigenic structure of HIV, to establish and identify immunologic classifications for HIV, and to choose rationally the minimum number and types of viruses from these immunologic groupings that will induce the broadest protective responses.

AIDS Vaccines↗
PubMed2001

Genotyping system of GBV-C/HGV type 1 to type 4 by the polymerase chain reaction using type-specific primers and geographical distribution of viral genotypes.

Based on variation in nucleotide sequence of 5'-untranslated region, GB virus type C (GBV-C) and hepatitis G virus (HGV) can be classified into three major genotypes. In addition to this classification, a novel genotype of GBV-C/HGV was identified and designated type 4. However, genotyping of GBV-C/HGV has been hampered by the lack of suitable assays. In this study, a simple and precise genotyping system based on PCR using the type-specific primers was developed for the determination of genotypes 1, 2, 3, and 4 of GBV-C/HGV. A total of 235 serum samples obtained from 11 different countries were tested by our PCR genotyping system of GBV-C/HGV. The results revealed that type 1 was prevalent mainly in Ghana, type 2 was prevalent in the USA, Spain, Egypt, Nepal and Myanmar, type 3 was prevalent in Japan and Bolivia, and type 4 was prevalent in Vietnam and Myanmar among the countries investigated in the present study. To confirm the specificity of the results of genotyping by PCR, phylogenetic analysis in the 5'-untranslated region of GBV-C/HGV was undertaken in 99 serum samples. By this analysis, the specificity of the genotyping system by PCR was confirmed. This assay system may be useful for rapid typing of GBV-C/HGV RNA when either epidemiological or transmission studies of this agent are carried out.

DNA Primers↗
PubMed2001

Clinically relevant sequence-based genotyping of HBV, HCV, CMV, and HIV.

The term 'genotyping' describes the genetic characterization of a genome. The genotype analysis is performed to identify mutations that differentiate one individual or strain from another. The mutations may confer resistance to specific antiviral drugs or they may simply allow classification of a strain as to 'type' and 'subtype'. There are four human viruses for which genotype information is clinically useful. Hepatitis B virus (HBV) infections are being treated with antiretroviral drugs and resistance after prolonged treatment is common. Since HBV cannot be cultured, the only method of detecting resistance-conferring mutations in the genome is a genotypic analysis. Hepatitis C virus (HCV) infection can be cured by treatment with the combination of interferon and ribavirin but certain strains of virus are more resistant to treatment than others. The current recommendations are that all HCV type 1 infections be treated for 12 months whereas other types may be successfully treated in 6 months. Since interferon treatment may have significant side effects, the determination of HCV genotype is an important aspect of this therapeutic regimen. Treatment of cytomegalovirus (CMV) disease with nucleoside analogues occasionally results in resistant virus with mutations in the phosphotransferase gene (UL97) and/or the DNA polymerase gene (UL54) that can be tested with phenotypic or genotypic assays. Since CMV grows very slowly, it may be more clinically useful to perform a rapid genotypic assay although only the UL97 gene can be efficiently genotyped. Finally, the virus for which genotyping has become the standard of care, human immunodeficiency virus type 1 (HIV-1) can now be genotyped routinely by many clinical virology labs experienced with molecular amplification methods and automated DNA sequencing technology. All currently-available antiretroviral drugs are directed against either the protease or reverse transcriptase genes of HIV-1 and the mutations within these genes that confer resistance have been well described. Sequence-based genotyping methods are not necessarily the best approach for routine genotyping of these four viruses, but sequencing is the gold standard from which other methods are developed and against which they are compared.

Base Sequence↗