RESUMO
Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.
RESUMO
Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Demência Frontotemporal/fisiopatologia , Modelos Teóricos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estresse Fisiológico , Linhagem Celular , Sobrevivência Celular , Humanos , Optogenética/métodos , Estimulação LuminosaRESUMO
Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain of function of the androgen receptor (AR). Previously, we found that co-regulator binding through the activation function-2 (AF2) domain of AR is essential for pathogenesis, suggesting that AF2 may be a potential drug target for selective modulation of toxic AR activity. We screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice revealed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain and spinal cord. In a preclinical trial in a new mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.
Assuntos
Atrofia Muscular Espinal/patologia , Degeneração Neural/patologia , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Animais , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Proteínas Correpressoras/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Atrofia Muscular Espinal/tratamento farmacológico , Degeneração Neural/tratamento farmacológico , Fenótipo , Projetos Piloto , Domínios Proteicos , Expansão das Repetições de Trinucleotídeos/genética , ortoaminobenzoatos/farmacologia , ortoaminobenzoatos/uso terapêuticoRESUMO
Using cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry and CLIP-Seq sequencing, we determined the peptide and oligonucleotide sequences at the interfaces between the capsid proteins and the genomic RNA of bacteriophage MS2. The results suggest that the same coat protein (CP)-RNA and maturation protein (MP)-RNA interfaces are used in every viral particle. The portions of the viral RNA in contact with CP subunits span the genome, consistent with a large number of discrete and similar contacts within each particle. Many of these sites match previous predictions of the locations of multiple, dispersed and degenerate RNA sites with cognate CP affinity termed packaging signals (PSs). Chemical RNA footprinting was used to compare the secondary structures of protein-free genomic fragments and the RNA in the virion. Some PSs are partially present in protein-free RNA but others would need to refold from their dominant solution conformations to form the contacts identified in the virion. The RNA-binding peptides within the MP map to two sections of the N-terminal half of the protein. Comparison of MP sequences from related phages suggests a similar arrangement of RNA-binding sites, although these N-terminal regions have only limited sequence conservation. In contrast, the sequences of the C-termini are highly conserved, consistent with them encompassing pilin-binding domains required for initial contact with host cells. These results provide independent and unambiguous support for the assembly of MS2 virions via a PS-mediated mechanism involving a series of induced-fit viral protein interactions with RNA.
Assuntos
Levivirus/fisiologia , Montagem de Vírus , Sítios de Ligação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Levivirus/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5' untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing â¼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3' portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.
Assuntos
Hepacivirus/metabolismo , Hepatócitos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , Sítios de Ligação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Hepacivirus/genética , Hepatócitos/patologia , Hepatócitos/virologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Fígado/patologia , Fígado/virologia , MicroRNAs/genética , Anotação de Sequência Molecular , Análise Serial de Proteínas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Transdução de Sinais , Replicação Viral/genéticaRESUMO
RNA nanotechnology often involves protein-RNA complexes that require significant understanding of how the proteins and RNAs contact each other. The CLIP-Seq (cross-linking immunoprecipitation, and DNA sequencing) protocol can be used to probe the RNA molecules that interact with proteins. We have optimized the procedures for RNA fragmentation, immunoprecipitation, and library construction in CLIP-Seq to map the interactions between the RNA and the capsid of a simple positive-strand RNA virus. The results show that distinct portions of the viral RNA contact the capsid. The protocol should be applicable to other RNA virions and also RNA-protein nanoparticles.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , Proteínas de Ligação a RNA/genética , Proteínas do Capsídeo/genética , Mapeamento de Interação de Proteínas/métodos , RNA Viral/genética , Vírion/genéticaRESUMO
BACKGROUND: Cisplatin, a platinum-derived chemotherapeutic agent, produces antineoplastic effects coupled with toxic neuropathic pain and impaired general health status. These side-effects complicate long term studies of neuropathy or analgesic interventions in animals. We recently demonstrated that pretreatment with sodium bicarbonate (4% NaHCO3) prior to cisplatin (3 mg/kg i.p. weekly up to 5 weeks) was associated with improved health status (i.e. normal weight gain, body temperature, creatinine and ketone levels, and kidney weight ratio) in rats (Neurosci Lett 544:41-46, 2013). To reduce the nephrotoxic effects of cisplatin treatment in mice, we compared effects of sodium bicarbonate (4% NaHCO3 s.c.), vitamin C (25 mg/kg s.c.), resveratrol (25 mg/kg s.c.) and saline (0.9% NaCl) pretreatment on cisplatin-induced changes in animal health status, neuropathic pain and proinflammatory cytokine levels in spinal cord and kidney. RESULTS: Cisplatin-treated mice receiving saline pretreatment exhibited elevated ketone, creatinine and kidney weight ratios, representative of nephrotoxicity. Vitamin C and sodium bicarbonate lowered creatinine/ketone levels and kidney weight ratio whereas resveratrol normalized creatinine levels and kidney weight ratios similar to saline pretreatment. All pretreatments were associated with decreased ketone levels compared to saline pretreatment. Cisplatin-induced neuropathy (i.e. mechanical and cold allodynia) developed equivalently in all pretreatment groups and was similarly reversed by either morphine (6 mg/kg i.p.) or ibuprofen (6 mg/kg i.p.) treatment. RT-PCR showed that mRNA levels for IL-1ß were increased in lumbar spinal cord of cisplatin-treated groups pretreated with either saline, NaHCO3 or resveratrol/cisplatin-treated groups. However, IL-6 and TNF-alpha were elevated in the kidneys in all cisplatin-treated groups. Our studies also demonstrate that 60 days after the last cisplatin treatment, body weight, body temperature, kidney functions and mRNA levels have returned to baseline although the neuropathic pain (mechanical and cold) is maintained. CONCLUSIONS: Studies employing cisplatin should include NaHCO3 or vitamin C pretreatment to improve animal health status and reduce nephrotoxicity (lower creatinine and kidney weight ratio) without affecting the development of chemotherapy-induced neuropathy or analgesic efficacy.
Assuntos
Ácido Ascórbico/administração & dosagem , Nível de Saúde , Doenças do Sistema Nervoso Periférico/prevenção & controle , Bicarbonato de Sódio/administração & dosagem , Vitaminas/administração & dosagem , Animais , Antineoplásicos/toxicidade , Glicemia/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cisplatino/toxicidade , Creatinina/sangue , Modelos Animais de Doenças , Esquema de Medicação , Hiperalgesia/induzido quimicamente , Hiperalgesia/prevenção & controle , Cetonas/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Limiar da Dor/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/sangue , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológicoRESUMO
Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of â¼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.
Assuntos
Hepacivirus/genética , Hepatite/virologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , RNA Viral/genética , Regulação Viral da Expressão Gênica , Hepacivirus/patogenicidade , Hepatite/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Análise Serial de Proteínas , Proteoma , Proteínas de Ligação a RNA/genética , Replicação Viral/genéticaRESUMO
The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is essential for the processing of the HCV polyprotein, the replication of HCV RNA, and to short circuit innate immunity signaling. NS3 contains an N-terminal domain with protease activity and a C-terminal domain with helicase activity. The two domains communicate with each other along with other HCV and cellular proteins. Herein we show that RNAs can bind directly to the active site cleft of the NS3 protease domain (NS3P) and inhibit proteolysis of peptide substrates. RNAs that are less apt to form intramolecular structures have a stronger inhibitory activity than RNAs with more stable base paired regions. Two mutations in the protease domain that resulted in decreased affinity to ssRNA were also defective in RNA-induced ATPase activity from the helicase domain of NS3. The coordinated effects on inhibition of protease activity and stimulation of ATPase activity raise the possibility that RNA serves as a regulatory switch for the two processes.
Assuntos
Hepacivirus/enzimologia , RNA/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação , Hepacivirus/fisiologia , Modelos Moleculares , Ligação Proteica , Proteólise , Proteínas não Estruturais Virais/químicaRESUMO
Understanding how the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) interacts with nascent RNA would provide valuable insight into the virus's mechanism for RNA synthesis. Using a peptide mass fingerprinting method and affinity capture of peptides reversibly cross-linked to an alkyn-labeled nascent RNA, we identified a region below the Δ1 loop in the fingers domain of the HCV RdRp that contacts the nascent RNA. A modification protection assay was used to confirm the assignment. Several mutations within the putative nascent RNA binding region were generated and analyzed for RNA synthesis in vitro and in the HCV subgenomic replicon. All mutations tested within this region showed a decrease in primer-dependent RNA synthesis and decreased stabilization of the ternary complex. The results from this study advance our understanding of the structure and function of the HCV RdRp and the requirements for HCV RNA synthesis. In addition, a model of nascent RNA interaction is compared with results from structural studies.
Assuntos
Hepacivirus/enzimologia , RNA Viral/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas Virais/genéticaRESUMO
Lactobacillus-dominated vaginal microbiotas are associated with reproductive health and STI resistance in women, whereas altered microbiotas are associated with bacterial vaginosis (BV), STI risk and poor reproductive outcomes. Putative vaginal taxa have been observed in male first-catch urine, urethral swab and coronal sulcus (CS) specimens but the significance of these observations is unclear. We used 16 S rRNA sequencing to characterize the microbiota of the CS and urine collected from 18 adolescent men over three consecutive months. CS microbiotas of most participants were more stable than their urine microbiotas and the composition of CS microbiotas were strongly influenced by circumcision. BV-associated taxa, including Atopobium, Megasphaera, Mobiluncus, Prevotella and Gemella, were detected in CS specimens from sexually experienced and inexperienced participants. In contrast, urine primarily contained taxa that were not abundant in CS specimens. Lactobacilllus and Streptococcus were major urine taxa but their abundance was inversely correlated. In contrast, Sneathia, Mycoplasma and Ureaplasma were only found in urine from sexually active participants. Thus, the CS and urine support stable and distinct bacterial communities. Finally, our results suggest that the penis and the urethra can be colonized by a variety of BV-associated taxa and that some of these colonizations result from partnered sexual activity.
Assuntos
Metagenoma , Pênis/microbiologia , Uretra/microbiologia , Adolescente , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Circuncisão Masculina , Estudos de Coortes , Feminino , Humanos , Masculino , Metagenoma/genética , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Parceiros Sexuais , Urina/microbiologia , Vaginose Bacteriana/microbiologiaRESUMO
Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC(50)s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (K(d)) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.
Assuntos
Antivirais/farmacologia , Cicloexanóis/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Pironas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Tiofenos/farmacologia , Triazóis/farmacologia , Antivirais/metabolismo , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Cicloexanóis/metabolismo , Farmacorresistência Viral , Inibidores Enzimáticos/metabolismo , Hepacivirus/enzimologia , Humanos , Modelos Moleculares , Mutação/efeitos dos fármacos , Pironas/química , Pironas/metabolismo , RNA Viral/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Replicon/efeitos dos fármacos , Moldes Genéticos , Tiofenos/metabolismo , Triazóis/química , Triazóis/metabolismoRESUMO
BACKGROUND: The microbiome of the male urogenital tract is poorly described but it has been suggested that bacterial colonization of the male urethra might impact risk of sexually transmitted infection (STI). Previous cultivation-dependent studies showed that a variety of non-pathogenic bacteria colonize the urethra but did not thoroughly characterize these microbiomes or establish links between the compositions of urethral microbiomes and STI. METHODOLOGY/FINDINGS: Here, we used 16S rRNA PCR and sequencing to identify bacteria in urine specimens collected from men who lacked symptoms of urethral inflammation but who differed in status for STI. All of the urine samples contained multiple bacterial genera and many contained taxa that colonize the human vagina. Uncultivated bacteria associated with female genital tract pathology were abundant in specimens from men who had STI. CONCLUSIONS: Urine microbiomes from men with STI were dominated by fastidious, anaerobic and uncultivated bacteria. The same taxa were rare in STI negative individuals. Our findings suggest that the composition of male urine microbiomes is related to STI.
Assuntos
Bactérias/isolamento & purificação , Metagenoma , Infecções Sexualmente Transmissíveis/microbiologia , Urina/microbiologia , Adulto , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Infecções Sexualmente Transmissíveis/urina , Uretra/microbiologia , Adulto JovemRESUMO
BACKGROUND: In 2003, severe acute respiratory syndrome (SARS) resulted in hundreds of infections and deaths globally. We aim to assess immunogenicity and protective efficacy of purified inactivated Vero-cell SARS vaccine in monkeys. METHODS: The cultures of SARS coronavirus (SARS-CoV) BJ-01 strain infected Vero cells were inactivated with beta-propiolactone. Sequential procedures, including ultrafiltration, gel filtration and ion exchange chromatography, were performed to obtain purified inactivated SARS vaccine. The purified SARS vaccine was analyzed with electron microscope, HPLC and Western blotting. We immunized three groups of cynomolgus macaques fascicularis with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine, respectively, and a fourth group served as a control. Antibody titers were measured by plaque reduction neutralization test. The vaccinated monkeys were challenged with SARS-CoV BJ-01 strain to observe protective efficacy. Additionally, three groups of rhesus monkeys were immunized with different doses of the purified inactivated SARS vaccine (0.5, 1 and 2mug/time/monkey) on days 0 and 7, and the monkeys were challenged with SARS-CoV GZ-01 strain. We assessed the safety of the SARS vaccine and observed whether the antibody dependent enhancement (ADE) occurred under low levels of neutralizing antibody in rhesus. FINDINGS: The purity of SARS vaccine was 97.6% by HPLC identification and reacted with convalescent sera of SARS patients. The purified SARS vaccine induced high levels of neutralizing antibodies and prevented the replication of SARS-CoV in monkeys. Under low levels of neutralizing antibody, no exacerbation of clinical symptoms was observed when the immunized monkeys were challenged with SARS-CoV. In this preliminary animal trial, no side effects were detected when monkeys were immunized with purified SARS vaccine either at normal or large doses. INTERPRETATION: The purified inactivated SARS vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of SARS-CoV. The SARS vaccine prepared in the study appeared to be safe in monkeys.
Assuntos
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Relação Dose-Resposta Imunológica , Imunização , Macaca fascicularis , Masculino , Síndrome Respiratória Aguda Grave/prevenção & controle , Vacinas de Produtos Inativados/imunologia , Células Vero , Vacinas Virais/efeitos adversosRESUMO
AIM: To develop a diagnostic kit for detection of SARS (severe acute respiratory syndrome)-related coronavirus RNA based on reverse transcription and polymerase chain reaction and to estimate its specificity and sensitivity. MATERIAL AND METHODS: 68 virus and bacterial cultures, 240 clinical samples from people without SARS symptoms and also 22 RNA samples from patients with SARS symptoms received during the epidemic in Beijing were used. RESULTS: The specificity of the kit was determined using animal coronaviruses and other bacterial and viral strains, causing acute respiratory and intestinal infections, and was shown to be 100%. The sensitivity of the kit in different clinical samples was 2.2 x 10(3) genome equivalents of recombinant SARS RNA in 1 ml of the specimen. The kit was evaluated in the Institute of Microbiology and Epidemiology of Beijing (China) using SARS-cov viral suspension and clinical samples from patients with suspected SARS. It was shown that kit was able to detect 10 TCID/50 ml of SARS-Cov virus. Testing of clinical samples from patients with suspected SARS showed that diagnostic sensitivity of the kit was 95%. Detection of the SARS-Cov RNA was more effective in feces compared to sputum 990 and 40%, respectively). CONCLUSION: The kit "AmpliSens SARS" for qualitative detection of SARS-related coronavirus RNA by reverse transcription and polymerase chain reaction (PCR) in nasopharyngeal wash/aspirates, naso/oropharyngeal swabs, plasma, and extract from feces has been developed in the Central Research Institute for Epidemiology of the RF Ministry of Health. The kit contains reagents for RNA isolation and purification, cDNA synthesis by reverse transcription of RNA, for PCR and for electrophoretic analysis of amplified products. The kit also contains recombinant positive and internal control samples allowing to control efficiency of analysis and showed good analytical and diagnostic characteristics.
Assuntos
RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Estudos de Avaliação como Assunto , Feminino , Humanos , Masculino , Sensibilidade e Especificidade , Síndrome Respiratória Aguda Grave/diagnóstico , Síndrome Respiratória Aguda Grave/patologia , Síndrome Respiratória Aguda Grave/virologiaRESUMO
IgG and IgM antibodies to dengue virus(DEN) in patient serum sample were detected with an indirect immunofluorescence assay. The patient's serum was inoculated into the culture of C6/36 cells to isolate dengue virus. Viral RNA was extracted from cultured supernatant and RT-PCR amplification and sequencing were carried out. The result showed that there were anti-dengue virus IgG and IgM antibodies in the serum. Both dengue type 2 and 3 virus specific sequences were confirmed by RT-PCR and sequence analysis. The results presented here demonstrated the simultaneous infection with dengue type 2 and 3 viruses in the Chinese patient.
Assuntos
Vírus da Dengue/classificação , Dengue/virologia , Adulto , Anticorpos Antivirais/sangue , Sequência de Bases , Efeito Citopatogênico Viral , Vírus da Dengue/isolamento & purificação , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
pDVWS501 was a genomic-length cDNA clone of dengue 2 virus, through which infectious virus (MON501) could be rescued. MON501 was neurovirulent in mice, whose E residues 62 and 203 were Lys and Asn, respectively. Two genomic-length cDNA clones (TB62 and TB203) were constructed by pointed mutation of pDVWS501 with OL-PCR, E62 of TB62 and E203 of TB203 were converted to Glu and Asp, respectively. RNA transcripts of pDVWS501, TB62 and TB203 were produced in vitro and electroporated into BHK-21 cells. The cultures were collected after 7 days and used as inoculum to infect C6/36 cells. The existence of rescued dengue viruses in the culture was proved by RT-PCR, and the typical cytopathic effect (CPE) of C6/36 caused by dengue virus emerged after 2-5 days' inoculation. Sequence analysis further confirmed the existence of recovered and recombinant DEN2 viruses, whose 5' termini had an additional non-virus nucleotides "G", while the 3' terminal sequences remained the same as natural. The neurovirulence of three viruses was evaluated in 1-day-old mice by the intracerebral route with 10(5)-10(2) TCID50. Compared with MON501 group, the number of infected mice with the signs of encephalitis in HFT62 and HFT203 groups was less, and the surviving time was longer. The properties of these mutants demonstrated that E62 and E203 are determinants of suckling mice neurovirulence.
Assuntos
Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Animais , Animais Lactentes , Sequência de Bases , Linhagem Celular , Cricetinae , Efeito Citopatogênico Viral/genética , Primers do DNA/genética , DNA Viral/genética , Dengue/virologia , Vírus da Dengue/classificação , Modelos Animais de Doenças , Encefalite Viral/virologia , Genes Virais , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Virulência/genéticaRESUMO
We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Assuntos
Variação Genética , Genoma Viral , Filogenia , Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Sequência de Bases , China , Análise por Conglomerados , Componentes do Gene , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.