RESUMEN
AIMS: Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. METHODS AND RESULTS: Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. CONCLUSIONS: Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. SIGNIFICANCE AND IMPACT OF THE STUDY: We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.
Asunto(s)
Bacterias/clasificación , ARN Ribosómico 16S/química , Análisis de Secuencia de ADN/métodos , Bacterias/aislamiento & purificación , Secuencia de Bases , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/instrumentaciónRESUMEN
The cytotoxic T-lymphocyte (CTL) response against the murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) 89-kDa phosphoprotein pp89 plays a major role in protecting BALB/c mice against the lethal effects of the viral infection. CTL populations specific to MCMV early-phase and structural antigens are also generated during infection, but the identities of these antigens and their relative contributions to overall immunity against MCMV are not known. We previously demonstrated that DNA vaccination with a pp89-expressing plasmid effectively generated a CTL response and conferred protection against infection (J. C. Gonzalez Armas, C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 70:7921-7928, 1996). In this report, we have sought (i) to identify other viral antigens that contribute to immunity against MCMV and (ii) to determine whether the protective response is haplotype specific. DNA immunization was used to test the protective efficacies of plasmids encoding MCMV homologs of human cytomegalovirus (HCMV) tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86), and nonstructural antigens (IE1-pp89 and M84). BALB/c (H-2(d)) and C3H/HeN (H-2(k)) mice were immunized by intradermal injection of either single plasmids or cocktails of up to four expression plasmids and then challenged with sublethal doses of virulent MCMV administered intraperitoneally. In this way, we identified a new viral gene product, M84, that conferred protection against viral replication in the spleens of BALB/c mice. M84 is expressed early in the infection and encodes a nonstructural protein that shares significant amino acid homology with the HCMV UL83-pp65 tegument protein, a major target of protective CTLs in humans. Specificity of the immune response to the M84 protein was confirmed by showing that immunization with pp89 DNA, but not M84 DNA, protected mice against subsequent infection with an MCMV deletion mutant lacking the M84 gene. The other MCMV genes tested did not generate a protective response even when mice were immunized with vaccinia viruses expressing the viral proteins. However, the M84 plasmid was protective when injected in combination with nonprotective plasmids, and coimmunization of BALB/c mice with pp89 and M84 provided a synergistic level of protection in the spleen. Viral titers in the salivary glands were also reduced, but not to the same extent as observed in the spleen, and the decrease was seen only when the BALB/c mice were immunized with pp89 plus M84 or with pp89 alone. The experiments with the C3H/HeN mice showed that the immunity conferred by DNA vaccination was haplotype dependent. In this strain of mice, only pp89 elicited a protective response as measured by a reduction in spleen titer. These results suggest that DNA immunization with the appropriate combination of CMV genes may provide a strategy for improving vaccine efficacy.
Asunto(s)
Infecciones por Herpesviridae/prevención & control , Muromegalovirus/inmunología , Muromegalovirus/fisiología , Vacunas de ADN/inmunología , Proteínas no Estructurales Virales/genética , Vacunas Virales/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Plásmidos/genética , Vacunación , Vacunas de ADN/administración & dosificación , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/metabolismo , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/administración & dosificación , Replicación ViralRESUMEN
We previously identified two open reading frames (ORFs) of murine cytomegalovirus (MCMV), M83 and M84, which are putative homologs of the human cytomegalovirus (HCMV) UL83 tegument phosphoprotein pp65 (L. D. Cranmer, C. L. Clark, C. S. Morello, H. E. Farrell, W. D. Rawlinson, and D. H. Spector, J. Virol. 70:7929-7939, 1996). In this report, we show that unlike the M83 gene product, the M84 protein is expressed at early times in the infection and cannot be detected in the virion. To elucidate the functional differences between the two pp65 homologs in acute and latent MCMV infections, we constructed two MCMV K181 mutants in which either the M83 or M84 ORF was deleted. The resultant viruses, designated DeltaM83 and DeltaM84, respectively, were found to replicate in NIH 3T3 cells with kinetics identical to those of the parent strain. Western blot analysis demonstrated that except for the absence of M83 or M84 protein expression in the respective mutants, no global perturbations of protein expression were detected. When DeltaM83 and DeltaM84 were inoculated intraperitoneally (i.p.) into BALB/c mice, both viruses showed similar attenuated growth in the spleen, liver, and kidney. However, only DeltaM83 was severely growth restricted in the salivary glands, a phenotype that was abolished upon restoration of the M83 ORF. DeltaM83's growth was similarly restricted in the salivary glands of the resistant C3H/HeN or highly sensitive 129/J strain, as well as in the lungs of all three strains following intranasal inoculation. Using a nested-PCR assay, we found that both DeltaM83 and DeltaM84 established latency in BALB/c mice, with slightly decreased levels of DeltaM83 and DeltaM84 genomic DNAs, relative to K181, observed in the salivary glands and lungs. Immunization of BALB/c mice with 10(5) PFU of K181, DeltaM83, or DeltaM84 i.p. provided similar levels of protection against lethal challenge. Although immunization with 200 PFU of DeltaM83 also provided complete protection, this dose allowed both the immunizing and challenge viruses to establish latency in the spleen. Our results show that the two MCMV pp65 homologs differ in their expression kinetics, virion association, and influence on viral tropism and/or dissemination.
Asunto(s)
Muromegalovirus/inmunología , Muromegalovirus/fisiología , Fosfoproteínas/fisiología , Proteínas de la Matriz Viral/fisiología , Latencia del Virus , Replicación Viral , Células 3T3 , Animales , Femenino , Eliminación de Gen , Infecciones por Herpesviridae/virología , Humanos , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Muromegalovirus/genética , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Glándulas Salivales/virología , Factores de Tiempo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunologíaRESUMEN
The murine cytomegalovirus (MCMV) immediate-early gene 1 (IE1) encodes an 89-kDa phosphoprotein (pp89) which plays a key role in protecting BALB/c mice against the lethal effects of the MCMV infection. In this report, we have addressed the question of whether "naked DNA" vaccination with a eukaryotic expression vector (pcDNA-89) that contains the MCMV IE1 gene driven by a strong enhancer/promoter can confer protection. BALB/c mice were immunized intradermally with pcDNA-89 or with the plasmid backbone pcDNAI/Amp (pcDNA) and then challenged 2 weeks later with either a lethal or a sublethal intraperitoneal dose of the K181 strain of MCMV. Variable results were obtained for the individual experiments in which mice received a lethal challenge. In four separate trials, an average of 63% of the mice immunized with pcDNA-89 survived, compared with 18% of the mice immunized with pcDNA. However, in two other trials there was no specific protection. The results of experiments in which mice were injected with a sublethal dose of MCMV were more consistent, and significant decreases in viral titer in the spleen and salivary glands of pcDNA-89-immunized mice were observed, relative to controls. At the time of peak viral replication, titers in the spleens of immunized mice were reduced 18- to >63-fold, while those in the salivary gland were reduced approximately 24- to 48-fold. Although DNA immunization elicited only a low level of seroconversion in these mice, by 7 weeks postimmunization the mice had generated a cytotoxic T-lymphocyte response against pp89. These results suggest that DNA vaccination with selected CMV genes may provide a safe and efficient means of immunizing against CMV disease.
Asunto(s)
Antígenos Virales/genética , ADN Viral/inmunología , Infecciones por Herpesviridae/prevención & control , Proteínas Inmediatas-Precoces/genética , Muromegalovirus/inmunología , Transactivadores/genética , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Células 3T3 , Animales , Anticuerpos Antivirales/inmunología , Células COS , Femenino , Inmunización , Ratones , Ratones Endogámicos BALB C , Glándulas Salivales/inmunología , Glándulas Salivales/patología , Glándulas Salivales/virología , Bazo/inmunología , Bazo/patología , Bazo/virología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
We have identified three open reading frames (ORFs) in murine cytomegalovirus (MCMV), designated M82, M83, and M84, which likely encode homologs of the human cytomegalovirus (HCMV) UL82 and UL83 matrix phosphoproteins. These ORFs, in the HindIII C fragment of MCMV, are colinear with the UL82, UL83, and UL84 ORFs of HCMV. M82 encodes a 598-amino-acid (aa) protein with homology to UL82, M83 encodes an 809-aa protein with homology to UL82 and UL83, and M84 encodes a 587-aa protein with homology to UL83 and UL84. Analysis of transcription by Northern (RNA) blotting indicated that the M82 and M83 ORFs are transcribed as 2.2- and 5-kb mRNAs, respectively, at 24 to 48 h postinfection (p.i.), while M84 is transcribed as a 6.9-kb mRNA only at 8 h p.i. All transcripts appear to terminate at the same position 3' of the M82 ORF. Of the products of the three ORFs, only M83 is strongly recognized by hyperimmune mouse serum. The M83 protein is a virion-associated phosphoprotein with an apparent molecular mass of 125 kDa. In MCMV-infected cells, it is detectable by Western blotting (immunoblotting) only at 48 h p.i. in the absence of phosphonoacetic acid, consistent with late gene expression. The M83 ORF is also expressed at high levels in cells infected by a recombinant vaccinia virus and yields a protein which is serologically cross-reactive and comigrates with the authentic MCMV protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Asunto(s)
Muromegalovirus/genética , Fosfoproteínas/genética , Proteínas de la Matriz Viral/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Secuencia de Bases , Citomegalovirus/metabolismo , ADN Viral , Desoxirribonucleasa HindIII/metabolismo , Evolución Molecular , Femenino , Expresión Génica , Vectores Genéticos , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Muromegalovirus/metabolismo , Sistemas de Lectura Abierta , Fosfoproteínas/metabolismo , Fosforilación , Filogenia , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transcripción Genética , Virus Vaccinia/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/genética , Virión/metabolismoRESUMEN
We have identified, characterized, and expressed in bacteria and recombinant vaccinia viruses a protein which likely represents the murine cytomegalovirus (MCMV) homologue of the human cytomegalovirus (HCMV) 28-kDa matrix phosphoprotein, the product of the HCMV UL99 open reading frame (ORF). This protein, referred to as the MCMV UL99, is encoded by a 336-nucleotide ORF within the HindIII G fragment of MCMV strain Smith (K181). Using a DNA probe that corresponded to the amino terminus of the ORF, we detected a transcript of 4.8 kb at 8 hr and additional transcripts of 0.88, 2.4, and 5.7 kb at 24-48 hr postinfection (p.i.) of NIH 3T3 cells with MCMV. The smallest transcript is unspliced, initiates 235 nucleotides upstream from the start of the ORF, and utilizes a polyadenylation site located 62 nucleotides downstream from the end of the ORF. The ORF encodes a protein of 112 amino acids, with a predicted mass of 11.8 kDa. Comparison of the derived amino acid sequence with that of the HCMV UL99 gene product reveals 34.8% identity in an overlap of 66 amino acids. Within the amino acid sequence are at least two potential protein kinase C and one potential casein kinase II target motifs for phosphorylation. The ORF was cloned into the pGEX-KG prokaryotic expression vector and bacterially expressed protein was used to generate a specific rabbit antiserum against the protein. Western blotting of MCMV-infected NIH 3T3 cells showed that the ORF was expressed as a doublet of 16.3 and 15.2 kDa at 48 hr p.i. only in the absence of phosphonoacetic acid, thus demonstrating that this protein is a member of the true late gene class. The immunoreactive protein in MCMV-infected cells comigrated with that produced in cells infected with recombinant vaccinia virus containing the ORF. The protein appears to be part of the MCMV virion, is phosphorylated in vivo, and generates a strong humoral immune response following MCMV infection of BALB/c mice.
Asunto(s)
Antígenos Virales/genética , Muromegalovirus/genética , Proteínas Virales/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/biosíntesis , Antígenos Virales/inmunología , Secuencia de Bases , Clonación Molecular , Femenino , Vectores Genéticos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Muromegalovirus/inmunología , Fosforilación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Transcripción Genética , Virus Vaccinia , Proteínas Virales/biosíntesis , Proteínas Virales/inmunologíaRESUMEN
The circadian clock in the unicellular alga Gonyaulax polyedra is accelerated by a substance in extracts from the cells themselves. The extracts have been fractionated using the circadian rhythm of bioluminescence as bioassay. The active substance, termed gonyauline, has been isolated and characterized as a novel low molecular weight cyclopropanecarboxylic acid (S-methyl-cis-2-(methylthio) cyclopropanecarboxylic acid). Synthetic gonyauline has a similar shortening effect on the period of the circadian clock.