RESUMO
We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.
Assuntos
Biologia Computacional , Drosophila melanogaster/genética , Genoma , Análise de Sequência de DNA , Algoritmos , Animais , Cromatina/genética , Mapeamento de Sequências Contíguas , Eucromatina , Genes de Insetos , Heterocromatina/genética , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Sequências Repetitivas de Ácido Nucleico , Sitios de Sequências RotuladasRESUMO
A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
Assuntos
Genoma Humano , Projeto Genoma Humano , Análise de Sequência de DNA , Algoritmos , Animais , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Biologia Computacional , Sequência Consenso , Ilhas de CpG , DNA Intergênico , Bases de Dados Factuais , Evolução Molecular , Éxons , Feminino , Duplicação Gênica , Genes , Variação Genética , Humanos , Íntrons , Masculino , Fenótipo , Mapeamento Físico do Cromossomo , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Proteínas/fisiologia , Pseudogenes , Sequências Repetitivas de Ácido Nucleico , Retroelementos , Análise de Sequência de DNA/métodos , Especificidade da EspécieRESUMO
The replication of feline immunodeficiency virus (FIV) in cultured cells was inhibited by 2',3'-dideoxyadenosine (ddA) and by 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with IC50 values of 0.98 and 0.95 microM, respectively. The effects of the presumed active forms of these inhibitors, ddATP and PMEA-diphosphate (PMEApp), upon the FIV reverse transcriptase (RT) were examined with two different template-primer systems. Both of these compounds were potent inhibitors of the FIV RT in reactions with primed phi X-174 DNA, yielding Ki values of 8.8 nM for ddATP and 5.0 nM for PMEApp. However, they were both poor inhibitors of the reaction with poly(rU)-oligo(dA); concentrations of ddATP or PMEApp greater than 10 microM were required to inhibit this reaction by 50%. Further analysis of the reaction with poly(rU)-oligo(dA) revealed that even in the absence of inhibitors the primers were extended by less than 20 nucleotides. In contrast, high molecular weight products were obtained in reactions with phi X-174 DNA. These results suggest that the reaction of FIV RT with poly(rU)-oligo(dA) is not highly processive. The high degree of termination encountered during this reaction with poly(rU)-oligo(dA) may be responsible for the low inhibitory potential of ddATP and PMEApp.
Assuntos
Adenina/análogos & derivados , Antivirais/farmacologia , Didesoxiadenosina/farmacologia , Vírus da Imunodeficiência Felina/enzimologia , Organofosfonatos , Inibidores da Transcriptase Reversa , Adenina/farmacologia , Linhagem Celular , Células Cultivadas , Vírus da Imunodeficiência Felina/isolamento & purificação , Cinética , Moldes GenéticosRESUMO
Three groups of specific pathogen-free (SPF) domestic cats, each containing 5 animals, were infected with one of three closely related FIV variants and monitored for 36 weeks. A fourth group of 5 cats was sham-infected and served as uninfected controls. FIV variants included: (1) a fully virulent animal passaged FIV-Petaluma; (2) a Crandell feline kidney (CrFK) cell-adapted FIV-Petaluma (FIV-CrFK); and (3) a variant of FIV-CrFK (FIV-CrFKAZT) that had been selected in vitro for resistance to azidothymidine. Cats infected with fully virulent FIV-Petaluma strongly seroconverted, became persistently viremic, and exhibited lymphadenopathy, neutropenia, and inversion of the CD4+:CD8+ T cell ratio. Cats infected with FIV-CrFK seroconverted but the antibody responses were much weaker and more variable; two of the cats became transiently viremic and no hematologic abnormalities or clinical signs of illness other than a very mild lymphadenopathy were observed. None of the five cats inoculated with FIV-CrFKAZT seroconverted, became viremic, or exhibited any gross or hematologic signs of disease, even though proviral DNA was transiently detected in tissue following inoculation. This study demonstrates that the FIV infection model can be used to assess differences in the virulence of FIV variants, including variants selected for antiretroviral drug resistance.
Assuntos
Vírus da Imunodeficiência Felina/efeitos dos fármacos , Vírus da Imunodeficiência Felina/patogenicidade , Infecções por Lentivirus/veterinária , Animais , Antivirais/farmacologia , Sequência de Bases , Gatos , DNA Viral/análise , Modelos Animais de Doenças , Resistência Microbiana a Medicamentos , Vírus da Imunodeficiência Felina/isolamento & purificação , Infecções por Lentivirus/microbiologia , Infecções por Lentivirus/fisiopatologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , VirulênciaRESUMO
Toxic-shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus MN8 was grown in dialyzed beef heart infusion broth, or dialyzed beef heart infusion broth supplemented with 5% human blood or with 5% human semen, in the presence and absence of Today contraceptive sponges. Early growth of the organism was inhibited by the presence of the sponges in aerobic, static aerobic, and anaerobic cultures. In aerobic cultures that contained the sponge, minimal TSST-1 was detected by immunodiffusion assay, whereas in control cultures that did not contain the sponge, extensive toxin production was seen. In static and anaerobic cultures, the presence of the contraceptive sponge resulted in toxin levels less than or equal to the low levels seen in control cultures. These results suggest that Today contraceptive sponges may inhibit TSST-1 production by S aureus.
Assuntos
Toxinas Bacterianas , Dispositivos Anticoncepcionais Femininos/efeitos adversos , Enterotoxinas/biossíntese , Staphylococcus aureus/crescimento & desenvolvimento , Superantígenos , Aerobiose , Anaerobiose , Animais , Bovinos , Meios de Cultura/metabolismo , Depressão Química , Humanos , Concentração de Íons de Hidrogênio , Masculino , Choque Séptico/etiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismoRESUMO
A single dose of bacterial lipopolysaccharide, administered 21 days after mice had been treated with the macrophage toxins carrageenan, or microparticulate crystalline silica, resulted in a secondarytype antibody response. The phenomenon of pseudosecondary responsiveness was investigated to determine the mechanism for its generation. The results showed that generating a pseudosecondary response was T cell dependent; but except for this, it followed the same kinetic and genetic patterns as a true, two dose of antigens, secondary response. It was concluded that pseudosecondary responsiveness resulted from priming the mice by the consequential effects of the macrophage toxins; thereafter, secondary responsiveness to LPS was generated in a normal manner.
Assuntos
Antígenos de Bactérias/imunologia , Carragenina/farmacologia , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Dióxido de Silício/farmacologia , Dióxido de Tório/farmacologia , Animais , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos , Escherichia coli/imunologia , Camundongos , Camundongos Nus/imunologia , Linfócitos T/imunologiaRESUMO
We have developed a host range system to measure the mutation frequency of feline immunodeficiency virus (FIV), the feline homologue of human immunodeficiency virus type 1 (HIV-1). When wild-type FIV was grown in the presence of a known mutagen, 5-bromo-2'-deoxyuridine (BUdR), a dose-dependent increase of host range mutants was detected. Using this system, we have evaluated the effects of antiviral drugs upon the mutation frequency of FIV. Subinhibitory concentrations of 3'-azido-3'-deoxythymidine (AZT), the most common antiviral drug used in AIDS chemotherapy, increased the mutation frequency of FIV in a dose-dependent manner. Two other antivirals, 2',3'-dideoxyinosine (ddI) and 2'3'-dideoxycytidine (ddC), did not show this effect.
Assuntos
Antivirais/farmacologia , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Mutação/efeitos dos fármacos , Zidovudina/farmacologia , Animais , Bromodesoxiuridina/farmacologia , Gatos , Linhagem Celular , Didanosina/farmacologia , Relação Dose-Resposta a Droga , Frequência do Gene/efeitos dos fármacos , Vírus da Imunodeficiência Felina/genética , Rim/citologia , Rim/virologia , Cultura de Vírus , Zalcitabina/farmacologiaRESUMO
We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis.
Assuntos
Fármacos Anti-HIV/farmacologia , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Vírus da Imunodeficiência Felina/genética , Estavudina/farmacologia , Animais , Gatos , Linhagem Celular , Primers do DNA , DNA Viral/análise , DNA Viral/efeitos dos fármacos , DNA Viral/isolamento & purificação , Resistência a Medicamentos , Vírus da Imunodeficiência Felina/patogenicidade , Cinética , Mutagênese Sítio-Dirigida , Mapeamento por Restrição , Inibidores da Transcriptase Reversa/farmacologia , Ensaio de Placa ViralRESUMO
We selected 3'-azido-3'-deoxythymidine (AZT)-resistant mutants of feline immunodeficiency virus (FIV) in a cat cell culture system. The characterization of one of these mutants was facilitated by the development of a focal immunoassay which could accurately measure FIV infectivity. This assay was used to quantitate the susceptibility of FIV to various inhibitors. The AZT-resistant mutant was found to be cross-resistant to 3'-azido-2',3'-dideoxyuridine and 3'-azido-2',3'-dideoxyguanosine but remained sensitive to several other inhibitors (2',3'-dideoxyinosine, 2',3'-dideoxy-2',3'-didehydrothymidine, and phosphonoformate). These patterns of cross-resistance and sensitivity were similar to those of the AZT-resistant human immunodeficiency virus (HIV) that has recently been isolated from patients with AIDS (B. A. Larder and S. D. Kemp, Science 246:1155-1158, 1989). Like the AZT-resistant HIV, purified reverse transcriptase from mutant FIV failed to show resistance to the 5'-triphosphate of AZT. This mutant can be used in the FIV model system to study the mechanisms of drug resistance and to determine the pathogenicity of AZT-resistant mutants.
Assuntos
Antivirais/farmacologia , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Mutação , Zidovudina/farmacologia , Animais , Gatos , Linhagem Celular , Resistência Microbiana a Medicamentos , Vírus da Imunodeficiência Felina/genética , CinéticaRESUMO
We have selected and plaque purified zidovudine (3'-azido-3'-deoxythymidine [AZT])-resistant mutants from an infectious molecular clone of feline immunodeficiency virus (FIV). The patterns of cross-resistance and drug susceptibilities of these mutants were similar to those of the AZT-resistant FIV that we previously selected in vitro from a wild-type FIV population and to those of the most common AZT-resistant clinical isolates of human immunodeficiency virus type 1. Two AZT-resistant mutants of FIV, one selected from a normal population and one selected from the molecular clone, each reverted rapidly to an AZT-sensitive phenotype when passaged in the absence of drug. Sequence analysis of the reverse transcriptase (RT)-encoding region from the plaque-purified AZT-resistant FIV revealed a single base change at position 2939, resulting in a Glu-to-Lys substitution at amino acid 202 of the RT. Similar analyses of plaque-purified revertants showed that the phenotypic reversion was not the result of a genotypic reversion at this position and that no additional mutations existed within the RT-encoding region of the revertants. Moreover, RTs purified from the mutant and revertant were both resistant to the 5'-triphosphate of AZT. These results indicate the complexity of AZT resistance and suggest the presence of additional factors, outside the RT-encoding region, which may contribute to AZT resistance.
Assuntos
Vírus da Imunodeficiência Felina/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/genética , Zidovudina/farmacologia , Animais , Sequência de Bases , Gatos , Células Cultivadas , Clonagem Molecular , Resistência Microbiana a Medicamentos , Vírus da Imunodeficiência Felina/enzimologia , Vírus da Imunodeficiência Felina/genética , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , DNA Polimerase Dirigida por RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Seleção Genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Ensaio de Placa ViralRESUMO
Mutants of feline immunodeficiency virus (FIV) were selected in cell culture in the continuous presence of 10 microM (each) 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI). These mutants (AIR-1 and AIR-3) displayed a 13-fold resistance to AZT but had less than a 2-fold decrease in susceptibility to ddI. Interestingly, the AIR mutants were cross-resistant to phosphonoformate (PFA) and were hypersensitive to 2',3'-dideoxycytidine (ddC). Mutants of FIV were also selected in the presence of 10 microM ddI alone (DIS-1, DIS-2c), and these displayed a two- to fourfold decrease in susceptibility to ddI. Like the mutants selected with the combination of AZT plus ddI, DIS-1 and DIS-2c were cross-resistant to PFA and were hypersensitive to ddC. However, they remained as susceptible as wild-type FIV to AZT. Thus, the mutants selected with the combination of AZT plus ddI have phenotypes which reflect those obtained by selection with these drugs individually.
Assuntos
Antivirais/farmacologia , Didanosina/farmacologia , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Zidovudina/farmacologia , Animais , Gatos , Células Cultivadas , Resistência Microbiana a Medicamentos , Foscarnet/farmacologia , Vírus da Imunodeficiência Felina/genética , Mutação , Fenótipo , Zalcitabina/farmacologiaRESUMO
The timing and character of opening and closing temporomandibular joint sounds were evaluated in a sample of 33 asymptomatic subjects with reciprocal clicking. Using stereophonic headphones and videotape recordings, three consecutive reciprocal sounds were recorded and analyzed. The timing of the three consecutive opening and closing sounds was consistent in the majority of subjects, and occurred generally on late opening and middle to late closing. When the timing, character (frequency, amplitude, and duration), and wave patterns were evaluated, a notable amount of variation was observed in the overall sample. This suggested a variety of intra-articular reasons for the reciprocal sounds in this particular sample of subjects.
Assuntos
Auscultação , Transtornos da Articulação Temporomandibular/diagnóstico , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Som , Gravação em VídeoRESUMO
The sensitivities of reverse transcriptases (RTs) from feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV) were directly compared. The two enzymes had similar sensitivities to three analogs of dTTP, namely, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate. Each of these analogs demonstrated competitive inhibition of both enzymes. Ki values for inhibition of FIV RT by these three inhibitors were 3.3, 6.7, and 1.8 nM, respectively; Ki values for inhibition of the HIV enzyme were 6.5, 5.9, and 8.3 nM, respectively. Ratios of the Ki for the inhibitor to the Km for the substrate were also determined for each inhibitor, and no differences between the two enzymes greater than threefold were observed. Inhibition constants for 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were determined for FIV RT, and these were similar to published values for HIV RT. The activities of three dideoxynucleoside 5'-triphosphates against FIV RT were determined; ddGTP was slightly more potent than ddTTP, whereas both were much more effective than ddCTP. The activity of a noncompetitive inhibitor, phosphonoformate, was also examined with the FIV enzyme; it was much more active with poly(rA)-oligo(dT) as the template-primer than with poly(rC)-oligo(dG) or poly(rI)-oligo(dC).
Assuntos
HIV/enzimologia , Retroviridae/enzimologia , Inibidores da Transcriptase Reversa , Animais , Gatos , Humanos , Cinética , Nucleotídeos de Timina/metabolismoRESUMO
We have previously developed an in vitro system that allows quantitative evaluation of the fidelity of transcription during synthesis on a natural template in the presence of all four nucleotides. Here, we have employed this system using a TAA ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase past an adenine residue adducted at the N6-position with (-)-anti-trans- or (+)-anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full-length run-off transcripts. The extent of bypass was found to be 32% for the (-)-anti-trans-isomer and 18% for the (+)-anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass synthesis of the (-)-anti-trans-isomer was elevated 11,000-fold and that of the (+)-anti-trans-isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template, three- and six-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell.
Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/síntese química , Bacteriófago T7/enzimologia , Adutos de DNA/síntese química , RNA Polimerases Dirigidas por DNA/metabolismo , Mutagênicos/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/química , Sequência de Bases , Adutos de DNA/química , Isomerismo , Moldes Genéticos , Transcrição Gênica , Proteínas ViraisRESUMO
BACKGROUND AND OBJECTIVES: Current manufacture of intravenous immunoglobulin (Gamimune N) uses four cold-ethanol precipitation steps and solvent-detergent treatment. Our objective was to design a new manufacturing process to maximize immunoglobulin G (IgG) purity, achieve robust viral safety, preserve all the biological activities of antibody and avoid unnecessary protein loss. MATERIALS AND METHODS: The new process combines multiple functions in single steps. Caprylate is added to precipitate non-IgG proteins and to inactivate enveloped viruses. Two successive anion-exchange columns are used to purify IgG and remove caprylate. The new product, IGIV-C (Gamunex, 10%) is formulated with glycine at 100 mg/ml IgG, pH 4.25. Vials are incubated for 21 days at 23-27 degrees C in a final virus-inactivation step. RESULTS: Compared with the process for production of Gamimune N, that for IGIV-C requires a shorter production time, achieves more robust virus inactivation, increases IGIV yield from plasma, improves physiological IgG subclass distribution (resulting in higher levels of IgG4), and improves purity, with lower levels of IgA (40 microg/ml), IgM (< 2 microg/ml) and albumin (< 20 microg/ml). Antibody binding, opsonization and protective activities are similar. CONCLUSIONS: Compared with the current commercial process, the new IGIV-C manufacturing process produces a more highly purified preparation that contains slightly higher levels of IgG4 and retains antibody activities required for clinical efficacy.
Assuntos
Desinfecção/métodos , Imunoglobulinas Intravenosas/normas , Vírus/isolamento & purificação , Animais , Infecções Bacterianas/tratamento farmacológico , Caprilatos/farmacologia , Precipitação Química , Cromatografia , Desinfecção/normas , Contaminação de Medicamentos/prevenção & controle , Indústria Farmacêutica/métodos , Indústria Farmacêutica/normas , Humanos , Imunoglobulinas Intravenosas/análise , Imunoglobulinas Intravenosas/farmacologia , Camundongos , Taxa de Sobrevida , Viroses/prevenção & controle , Viroses/transmissãoRESUMO
BACKGROUND AND OBJECTIVES: Ultraviolet-C (UVC) irradiation is a viral-inactivation method that was dismissed by many plasma fractionators as a result of the potential for protein damage and the difficulty in delivering uniform doses. A reactor with novel spiral flow hydraulic mixing was recently designed for uniform and controlled UVC treatment. The objective of this study was to investigate virus inactivation and protein recovery after treatment through the new reactor. MATERIALS AND METHODS: Virus- and mock-spiked Alpha1-proteinase inhibitor (Alpha1-PI) solutions were treated with UVC. The virus samples were assayed for residual infectivity and amplified by the polymerase chain reaction (PCR). The mock-spiked samples were assayed for protein integrity. RESULTS: Greater than 4 log10 of all test viruses were inactivated, regardless of the type of nucleic acid or presence of an envelope. Unlike previous studies, viruses with the smallest genomes were found to be those most sensitive to UVC irradiation, and detection of PCR amplicons > or = 2.0 kb was correlated to viral infectivity. Doses that achieved significant virus inactivation yielded recovery of > 90% protein activity, even in the absence of quenchers. CONCLUSIONS: The results demonstrate the effectiveness of UVC treatment, in the novel reactor, to inactivate viruses without causing significant protein damage, and confirm the utility of large PCR amplicons as markers for infectious virus.
Assuntos
Desinfecção/instrumentação , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação , Vírus/efeitos da radiação , Adenovírus Humanos/fisiologia , Adenovírus Humanos/efeitos da radiação , Animais , Linhagem Celular/virologia , Chlorocebus aethiops , Cricetinae , DNA Viral/genética , DNA Viral/isolamento & purificação , DNA Viral/efeitos da radiação , Temperatura Alta , Humanos , Orthoreovirus Mamífero 3/fisiologia , Orthoreovirus Mamífero 3/efeitos da radiação , Mesocricetus , Parvovirus B19 Humano/fisiologia , Parvovirus B19 Humano/efeitos da radiação , Plasma , Plasmídeos/efeitos da radiação , Reação em Cadeia da Polimerase , Albumina Sérica/efeitos da radiação , Vírus 40 dos Símios/fisiologia , Vírus 40 dos Símios/efeitos da radiação , Sindbis virus/fisiologia , Sindbis virus/efeitos da radiação , Replicação Viral/efeitos da radiação , Vírus/crescimento & desenvolvimento , alfa 1-Antitripsina/química , alfa 1-Antitripsina/efeitos da radiaçãoRESUMO
Variants of feline immunodeficiency virus (FIV) that possess a unique methionine-to-threonine mutation within the YMDD motif of reverse transcriptase (RT) were selected by culturing virus in the presence of inhibitory concentrations of (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC]. The mutants were resistant to (-)-FTC and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and additionally exhibited low-level resistance to 2',3'-dideoxycytidine (ddC). DNA sequence analysis of the RT-encoding region of the pol gene amplified from resistant viruses consistently identified a Met-to-Thr mutation in the YMDD motif. Purified RT from the mutants was also resistant to the 5'-triphosphate forms of 3TC, (-)-FTC, and ddC. Site-directed mutants of FIV were engineered which contain either the novel Met-to-Thr mutation or the Met-to-Val mutation seen in oxathiolane nucleoside-resistant HIV-1. Both site-directed mutants displayed resistance to 3TC, thus confirming the role of these mutations in the resistance of FIV to beta-L-3'-thianucleosides.
Assuntos
Antivirais/farmacologia , Resistência Microbiana a Medicamentos/genética , Vírus da Imunodeficiência Felina/enzimologia , Lamivudina/farmacologia , Metionina , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Treonina , Zalcitabina/análogos & derivados , Zalcitabina/farmacologia , Animais , Sítios de Ligação , Gatos , Linhagem Celular , Emtricitabina/análogos & derivados , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Ensaio de Placa ViralRESUMO
Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.
Assuntos
Doenças do Gato/microbiologia , Síndromes de Imunodeficiência/veterinária , DNA Polimerase Dirigida por RNA/metabolismo , Retroviridae/enzimologia , Animais , Gatos , Cromatografia DEAE-Celulose , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Síndromes de Imunodeficiência/microbiologia , Cinética , Peso Molecular , Peptídeo Hidrolases , Mapeamento de Peptídeos , Polirribonucleotídeos , DNA Polimerase Dirigida por RNA/isolamento & purificação , Moldes GenéticosRESUMO
Mutants of feline immunodeficiency virus (FIV) resistant to (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3'-azido-3'-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5'-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation.
Assuntos
Vírus da Imunodeficiência Felina/enzimologia , Lamivudina/farmacologia , Mutação Puntual , Prolina/genética , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa/farmacologia , Serina/genética , Zidovudina/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Gatos , Linhagem Celular , Interações Medicamentosas , Resistência Microbiana a Medicamentos/genética , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/isolamento & purificação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Prolina/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Análise de Sequência de DNA , Serina/metabolismo , Ensaio de Placa ViralRESUMO
We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis.