RESUMEN
Hepatitis C virus (HCV) genotype 4 (GT4) is genetically diverse with 17 confirmed and 4 provisional subtypes. In this report, HCV GT4-infected patient samples from Phase 2/3 clinical studies were analysed to characterize global demographics and genetic diversity of GT4 infection among patients treated with ombitasvir (OBV, NS5A inhibitor) plus paritaprevir/r (NS3/4A inhibitor codosed with ritonavir). Among 17 subtypes isolated from GT4-infected patients in the PEARL-I and AGATE-I studies, subtype prevalence by country of enrolment and country of origin suggested that subtypes 4a and 4d were likely circulating in Europe, while heterogeneous GT4 subtypes and a portion of GT4a detected in European and North American countries were likely due to immigration of HCV-infected patients from Africa. The distributions of birth cohort and race were also significantly different across GT4 subtypes 4a, 4d, and non-4a/4d. In addition, phylogenetic analyses of NS5A sequences revealed clustering within subtype 4a which segregated by the patient-reported country of origin and the presence of the L30R/S polymorphism. HCV NS5A sequences derived from GT4a-infected patients who originated from Europe and the United States clustered separately from sequences derived from patients who originated from Egypt, suggesting that genetically distinct strains of subtype 4a may be circulating globally. Finally, NS5A baseline polymorphisms were frequently detected at amino acid positions of interest for the inhibitor-class and OBV retained activity against 37 of 39 NS5A GT4 clinical isolates, with no impact on treatment outcome in the PEARL-I and AGATE-I studies.
Asunto(s)
Antivirales/administración & dosificación , Variación Genética , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Proteínas no Estructurales Virales/genética , Adulto , Anciano , Anciano de 80 o más Años , Anilidas/administración & dosificación , Carbamatos/administración & dosificación , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Análisis por Conglomerados , Ciclopropanos , Demografía , Europa (Continente)/epidemiología , Femenino , Hepacivirus/clasificación , Hepatitis C Crónica/epidemiología , Humanos , Lactamas Macrocíclicas , Compuestos Macrocíclicos/administración & dosificación , Masculino , Persona de Mediana Edad , Epidemiología Molecular , América del Norte/epidemiología , Filogenia , Prevalencia , Prolina/análogos & derivados , Ritonavir/administración & dosificación , Análisis de Secuencia de ADN , Sulfonamidas , Resultado del Tratamiento , ValinaRESUMEN
The efficacy and safety of an investigational combination of ombitasvir/paritaprevir/ritonavir (OBV/PTV/r) plus sofosbuvir (SOF) ± ribavirin (RBV) in patients with HCV genotype 2 or 3 infection with or without cirrhosis was evaluated. Patients with HCV genotype 3 infection without cirrhosis were randomized to receive OBV/PTV/r + SOF ± RBV for 12 weeks; OBV/PTV/r + SOF + RBV was administered to genotype 3-infected patients with cirrhosis for 12 weeks and to genotype 2-infected patients without cirrhosis for either 6 or 8 weeks. Efficacy was assessed by sustained virologic response [HCV RNA <25 IU/mL] 12 weeks post-treatment (SVR12). Safety was assessed in all treated patients. In patients with genotype 3 infection with or without cirrhosis treated with 12 weeks of OBV/PTV/r + SOF ± RBV, the overall SVR12 rate was 98% (50/51), with no virologic failures. Patients with genotype 2 infection treated with OBV/PTV/r + SOF + RBV had SVR12 rates of 90% (9/10) and 44% (4/9) following 8- and 6-week treatment durations, respectively; failure to achieve SVR12 for these patients was due to relapse without baseline or treatment-emergent resistance-associated substitutions. Thus, the investigational combination of OBV/PTV/r with SOF ± RBV was well tolerated and achieved high SVR rates with no virologic failures in patients with genotype 3 infection. Combining direct-acting antivirals with complementary mechanisms of action and different viral targets may be an effective treatment strategy that may allow for shorter durations of therapy.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis C/tratamiento farmacológico , Cirrosis Hepática/tratamiento farmacológico , Respuesta Virológica Sostenida , Adulto , Anciano , Anilidas/administración & dosificación , Anilidas/uso terapéutico , Antivirales/administración & dosificación , Carbamatos/administración & dosificación , Carbamatos/uso terapéutico , Ciclopropanos , Quimioterapia Combinada , Femenino , Genotipo , Hepacivirus/genética , Humanos , Lactamas Macrocíclicas , Cirrosis Hepática/virología , Compuestos Macrocíclicos/administración & dosificación , Compuestos Macrocíclicos/uso terapéutico , Masculino , Persona de Mediana Edad , Prolina/análogos & derivados , ARN Viral/sangre , Ribavirina/administración & dosificación , Ribavirina/uso terapéutico , Ritonavir/administración & dosificación , Ritonavir/uso terapéutico , Sofosbuvir/administración & dosificación , Sofosbuvir/uso terapéutico , Sulfonamidas , Resultado del Tratamiento , ValinaRESUMEN
Two overlapping sets of TT virus (TTV)-specific polymerase chain reaction primers were used to test for presence of TTV, which was found in approximately 10% of US volunteer blood donors, 13% of commercial blood donors, and 17% of intravenous drug abusers. The rate of TTV infection among US non-A, non-B, non-C, non-D, non-E hepatitis patients was only 2%. Among commercial blood donors and intravenous drug abusers, only 1%-3% of the TTV-positive individuals were coinfected with GB virus C (GBV-C), a parenterally transmitted virus. This suggests that GBV-C and TTV may have different routes of transmission. Comparison of the sensitivities of 2 TTV polymerase chain reaction (PCR) primer sets showed that the majority of samples were detected with only 1 of the 2 sets. Therefore, previous studies in which only a single PCR primer pair was used may have significantly underestimated the true prevalence of TTV.
Asunto(s)
Donantes de Sangre , Infecciones por Virus ADN/epidemiología , Virus ADN/aislamiento & purificación , Virus de Hepatitis/aislamiento & purificación , Hepatitis Viral Humana/epidemiología , Abuso de Sustancias por Vía Intravenosa/complicaciones , Cartilla de ADN , Infecciones por Virus ADN/complicaciones , Infecciones por Virus ADN/virología , Virus ADN/genética , ADN Viral/análisis , Flaviviridae/genética , Flaviviridae/aislamiento & purificación , Virus de Hepatitis/genética , Hepatitis Viral Humana/complicaciones , Hepatitis Viral Humana/virología , Humanos , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Sensibilidad y Especificidad , Estados Unidos/epidemiologíaRESUMEN
The recent isolation of a novel DNA virus from the serum of a Japanese patient (T.T.) has provided the latest possible candidate virus associated with cryptogenic hepatitis. In the present study, we report the complete nucleotide sequence of this virus (TTV) isolated from the serum of a West African. Based on PCR studies designed to amplify overlapping regions of the viral genome and sensitivity to digestion with mung bean nuclease, the viral genome is circular and negative stranded, and comprises 3,852 nt, which is 113 nt longer than the prototype isolate from Japan. Cesium chloride density gradient centrifugation demonstrated banding of the virus at 1.31-1.34 g/ml; filtration studies indicated that TTV had a particle size of 30-50 nm. These results suggest that the virus is similar to the Circoviridae, viruses known to infect plants and vertebrates (e. g., birds and swine); however, sequence similarity searches of available databases did not reveal identity between TTV and other viruses. Phylogenetic analyses of a 260-nt region from 151 globally distributed isolates demonstrated the existence of three major TTV genotypes. Several individuals at high risk for infection with parenterally transmitted viruses were infected with more than one genotype. There was no correlation between genotype and geographic origin. Finally, intravenous inoculation of TTV-positive human serum into chimpanzees demonstrated that TTV can be transmitted to primates; no biochemical or histological evidence for hepatitis was obtained. The distinct biophysical and molecular characteristics of TTV suggest that it is a member of a new family of viruses, which we have tentatively named the Circinoviridae.
Asunto(s)
Virus ADN/aislamiento & purificación , Genoma Viral , Hepatitis Viral Humana/virología , Circoviridae/clasificación , Circoviridae/genética , Virus ADN/clasificación , Virus ADN/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADNRESUMEN
A 315 amino acid recombinant segment of the GB virus C (GBV-C) E2 envelope glycoprotein (E2-315) was expressed and secreted from CHO cells. E2-315 was purified by affinity chromatography using a monoclonal antibody directed to a FLAG sequence genetically engineered onto the C terminus of the recombinant protein. The secreted protein had a molecular mass of 48-56 kDa and was shown to be N-glycosylated. Amino acid sequencing confirmed the expected N-terminal sequence. Purified E2-315 was used to develop an ELISA for detection of E2 antibodies in human sera. Antibodies to GBV-C E2 appeared to be directed toward conformational epitopes since human sera reactivity was detected in ELISA using native E2-315, but it was extremely weak or non-existent with denatured E2 protein. The use of an ELISA which can detect human GBV-C E2 antibodies will be important in further understanding of the clinical significance and epidemiology of GBV-C.
Asunto(s)
Flaviviridae/metabolismo , Anticuerpos Antihepatitis/sangre , Proteínas del Envoltorio Viral/biosíntesis , Secuencia de Aminoácidos , Animales , Células CHO , Cromatografía de Afinidad , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Flaviviridae/genética , Glicosilación , Hepatitis Viral Humana/diagnóstico , Hepatitis Viral Humana/epidemiología , Hepatitis Viral Humana/inmunología , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transfección , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
A 336-amino-acid segment of the GB virus C second envelope protein (E2) has been produced in BHK-21 cells using the Semliki Forest virus vector system. Secretion of this protein was facilitated by deletion of a hydrophobic region at the C-terminus that may represent the membrane anchoring domain. The E2 protein recovered from the culture supernatant exhibited a molecular mass of approximately 52 kDa, with the increase in size relative to the polyprotein backbone being contributed by N-linked glycosylation. A radioimmunoprecipitation assay using GBV-C E2 was developed to test for the presence of antibodies against this protein in human sera. The prevalence of antibodies to E2 was high among injection drug users and other individuals at risk for acquiring parenterally transmitted agents. There was a much higher percentage of anti-E2 seropositivity in GBV-C RT-PCR negative compared to GBV-C RT-PCR positive samples from these populations. In addition, serial samples from patients transfused with blood containing GBV-C showed seroconversion to anti-E2 positivity and loss of GBV-C viremia as measured by RT-PCR within 11 months of transfusion in five of seven individuals. Thus, this system provided a rapid means to identify GBV-C E2 as a useful antigen for the study of GBV-C exposure.
Asunto(s)
Flaviviridae/genética , Vectores Genéticos , Virus de los Bosques Semliki/genética , Pruebas Serológicas , Proteínas del Envoltorio Viral/genética , Secuencia de Bases , Biomarcadores , Flaviviridae/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Proteínas del Envoltorio Viral/sangre , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
BACKGROUND: GB virus C is a positive-strand RNA virus that is associated with hepatitis in humans. GB virus C bears some resemblance to hepatitis C virus in its genomic sequence and organization. However, unlike hepatitis C virus, an open reading frame possessing a complete core protein was not identified in the original isolate. METHODS: To verify the sequence at the 5'-end of the GB virus C genome, we amplified approximately 600 nucleotides from this region from 35 globally distributed individuals. The nucleotide sequences were translated in all possible reading frames and then examined for conserved motifs indicative of nucleocapsid or core-like peptides. RESULTS: Forty-two unique GB virus C sequences were obtained from the 35 individuals. The deduced amino acid sequences upstream of the putative E1 gene from each isolate varied in length and composition, such that a conserved core-like sequence was not apparent. No core-like sequences were evident in the other reading frames. There was, however, a single methionine codon held in common among all isolates, although it was located very near the presumed amino-terminus of the putative E1 protein. Further analysis of the sequences for their evolutionary relatedness demonstrated the existence of five GB virus C subtypes that demonstrated a significant correlation with geographic distribution. CONCLUSIONS: GB virus C differs from hepatitis C virus and GB virus B in that it does not encode a nucleocapsid or core protein. The existence of GB virus C subtypes emphasizes the importance of investigating the correlation between infecting subtype and the severity of liver disease and/or responsiveness to treatment of GB virus C-associated hepatitis.
Asunto(s)
Flaviviridae/genética , Genoma Viral , Población , Secuencia de Bases , Genotipo , Humanos , Datos de Secuencia Molecular , Nucleocápside/genética , FilogeniaRESUMEN
Among the three recently described GB viruses (GBV-A, GBV-B, and GBV-C), only GBV-C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV-C proteins, the prevalence of GBV-C RNA in human sera was studied using reverse transcription-polymerase chain reaction (RT-PCR). The prevalence of GBV-C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV-C was frequently detected in residents of West Africa, where the prevalence was > 10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV-C RNA. In addition, GBV-C RNA sequences were detected in individuals diagnosed with non-A-E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV-C RNA more than 1 year after the initial positive result.
Asunto(s)
Flaviviridae/aislamiento & purificación , Hepatitis Viral Humana/virología , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangre , Flaviviridae/genética , Flaviviridae/fisiología , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/epidemiología , Humanos , Viremia , Latencia del VirusRESUMEN
The genomes of two novel members of the Flaviviridae associated with GB agent hepatitis (GB viruses A and B) were cloned and sequenced recently. The genome of a third novel virus (GB virus C), related to but distinct from GB viruses A and B, has also been identified and characterized. Overlapping clones encompassing the large open reading frames of these three viruses have been expressed in E. coli as CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CKS) fusion proteins. Bacterial lysates were subjected to Western blot analyses using sera from GB agent-infected tamarins and human sera from various individuals with or "at risk" for non-A, non-B, non-C, non-D, non-E hepatitis. Antigenic regions were identified in the putative NS3, NS4, and NS5 proteins from all three viruses. An antigenic region was also identified in the putative core protein of GB virus B. Many of the clones identified originally as encoding antigenic proteins were quite large. To map these regions more narrowly, smaller overlapping clones were generated by polymerase chain reaction (PCR), expressed as recombinant CKS fusion proteins and tested by Western blot. Additionally, a lambda gt11 expression library was generated from infectious tamarin sera and immunoscreened. These studies have identified at least three epitopes in GB virus A, five epitopes in GB virus B and four epitopes in GB virus C.
Asunto(s)
Antígenos Virales/análisis , Epítopos/análisis , Flaviviridae/genética , Genoma Viral , Virus de Hepatitis/genética , Hepatitis Viral Animal/virología , Hepatitis Viral Humana/virología , Animales , Secuencia de Bases , ADN Viral/análisis , Flaviviridae/inmunología , Virus de Hepatitis/inmunología , Hepatitis Viral Animal/inmunología , Hepatitis Viral Humana/inmunología , Humanos , Datos de Secuencia Molecular , SaguinusRESUMEN
Recently, sequences from a novel virus, termed GB virus C (GBV-C), were identified in serum from several patients with cryptogenic hepatitis. In the present study, the nucleotide sequence of this virus has been extended to near-genome length. GBV-C encodes a putative single large polyprotein in which the structural proteins are positioned at the N-terminal end, with the non-structural proteins located at the C-terminal end. Amino acid sequence analysis of this large polyprotein reveals the presence of protease, helicase, and replicase motifs. Sequence alignments of the polyprotein followed by phylogenetic analyses suggest that GBV-C is a member of the Flaviviridae, most closely related to the recently described GB virus A.
Asunto(s)
Genoma Viral , Virus de Hepatitis/genética , Hepatitis Viral Humana/virología , Secuencia de Aminoácidos , Endopeptidasas/metabolismo , Flaviviridae/química , Flaviviridae/clasificación , Flaviviridae/genética , Virus de Hepatitis/química , Virus de Hepatitis/clasificación , Humanos , Datos de Secuencia Molecular , Filogenia , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Virales/metabolismoRESUMEN
Recently, sequences from a putative member of the Flaviviridae, GB virus C (GBV-C), were isolated from the serum of patients with cryptogenic hepatitis. These sequences were 83-99% identical at the nucleotide level. Because of the divergence between these GBV-C isolates, it is likely that the PCR-based detection assay yields false negatives, underestimating dramatically the true prevalence of GBV-C in human hepatitis. We report the design of a GBV-C consensus oligonucleotide primer pair that is superior to those originally described. These primers identify GBV-C sequences in cases of cryptogenic hepatitis, allowing a better estimation of the prevalence of this virus in human populations.
Asunto(s)
Cartilla de ADN , Infecciones por Flaviviridae/virología , Flaviviridae/aislamiento & purificación , Hepatitis Viral Humana/virología , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Secuencia de Consenso , ADN Viral/análisis , Humanos , Datos de Secuencia MolecularRESUMEN
The genomes of two positive-strand RNA viruses have recently been cloned from the serum of a GB agent-infected tamarin by using representational difference analysis. The two agent, GB viruses A and B (GBV-A and GBV-B, respectively), have genomes of 9,493 and 9,143 nucleotides, respectively, and single large open reading frames that encode potential polyprotein precursors of 2,972 and 2,864 amino acids, respectively. The genomes of these agents are organized much like those of other pestiviruses and flaviviruses, with genes predicted to encode structural and nonstructural proteins located at the 5' and 3' ends, respectively. Amino acid sequence alignments and subsequent phylogenetic analysis of the RNA-dependent RNA polymerases (RdRps) of GBV-A and GBV-B show that they possess conserved sequence motifs associated with supergroup II RNA polymerases of positive-strand RNA viruses. On the basis of similar analyses, the GBV-A- and GBV-B-encoded helicases show significant identity with the supergroup II helicases of positive-strand RNA viruses. Within the supergroup II RNA polymerases and helicases, GBV-A and GBV-B are most closely related to the hepatitis C virus group. Across their entire open reading frames, the GB agents exhibit 27% amino sequence identity to each other, approximately 28% identity to hepatitis C virus type 1, and approximately 20% identity to either bovine viral diarrhea virus or yellow fever virus. The degree of sequence divergence between GBV-A and GBV-B and other Flaviviridae members demonstrates that the GB agents are representatives of two new genera within the Flaviviridae family.
Asunto(s)
Flaviviridae/genética , Genoma Viral , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Helicasas/química , ADN Helicasas/genética , Flaviviridae/aislamiento & purificación , Flaviviridae/patogenicidad , Infecciones por Flaviviridae/sangre , Infecciones por Flaviviridae/virología , Hepatitis Viral Animal/sangre , Hepatitis Viral Animal/virología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Saguinus/virología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genéticaRESUMEN
Two viruses, GB virus A (GBV-A) and GB virus B (GBV-B), were recently identified in the GB hepatitis agent. Human sera containing antibodies that recognize GBV-A and/or GBV-B recombinant proteins were subjected to polymerase chain reaction studies with degenerate oligonucleotides capable of amplifying a segment of the putative helicase genes from GBV-A, GBV-B or hepatitis C virus. Novel sequences related to members of the Flaviviridae were identified in sera from 12 individuals including 4 individuals with hepatitis. The limited nucleotide sequence identity between GBV-A, GBV-B and HCV sequences suggests that a novel virus, tentatively named GB virus C, may be responsible for some cases of non-A, non-B, non-C, non-D, non-E hepatitis.
Asunto(s)
Flaviviridae/aislamiento & purificación , Virus de Hepatitis/aislamiento & purificación , Hepatitis Viral Humana/virología , Viremia/virología , Adulto , África Oriental/epidemiología , África Occidental/epidemiología , Secuencia de Aminoácidos , Anticuerpos Antivirales/sangre , Secuencia de Bases , Canadá/epidemiología , Comorbilidad , ADN Helicasas/genética , Femenino , Flaviviridae/genética , Flaviviridae/inmunología , Virus de Hepatitis/genética , Virus de Hepatitis/inmunología , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/epidemiología , Humanos , Masculino , Tamizaje Masivo , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Prevalencia , Factores de Riesgo , Alineación de Secuencia , Homología de Secuencia , Abuso de Sustancias por Vía Intravenosa/epidemiología , Abuso de Sustancias por Vía Intravenosa/virología , Proteínas no Estructurales Virales/genéticaRESUMEN
Two flavivirus-like genomes have recently been cloned from infectious tamarin (Saguinus labiatus) serum, derived from the human viral hepatitis GB strain, which is known to induce hepatitis in tamarins. In order to study the natural history of GB infections, further transmission studies were carried out in tamarins. Reverse-transcription-polymerase chain reaction and enzyme-linked immunosorbant assays were developed for the detection of RNA and antibodies associated with the two agents, GB virus-A and GB virus-B. The infectivity of both of these agents was demonstrated in tamarins to be filterable through a 0.1 micron filter. Two distinct genomes were identified in the serum of eight of the infected tamarins, while in four tamarins, the genomes were detected independently of each other. Although specific antibodies to the GB virus-B epitopes were detected in the serum of animals inoculated with both agents or GB virus-B alone, antibodies to putative epitopes specific to GB virus-A were not detected in any of the animals. All tamarins inoculated with serum containing GB virus-B exhibited an elevation in liver enzyme levels after inoculation. Elevations of serum liver enzyme levels did not occur when GB virus-A was the only agent detected in the serum. Infection with the original infectious tamarin inoculum conferred protection from reinfection with GB virus-B but not with GB virus-A.
Asunto(s)
Flavivirus/genética , Virus de Hepatitis/genética , Hepatitis Viral Animal/transmisión , Hepatitis Viral Humana/transmisión , Animales , Anticuerpos Antivirales/inmunología , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Flavivirus/aislamiento & purificación , Flavivirus/patogenicidad , Virus de Hepatitis/aislamiento & purificación , Virus de Hepatitis/patogenicidad , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/virología , Hepatitis Viral Humana/inmunología , Hepatitis Viral Humana/virología , Humanos , Hígado/enzimología , Macaca , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , SaguinusRESUMEN
A subtractive PCR methodology known as representational difference analysis was used to clone specific nucleotide sequences present in the infectious plasma from a tamarin infected with the GB hepatitis agent. Eleven unique clones were identified, seven of which were examined extensively. All seven clones appeared to be derived from sequences exogenous to the genomes of humans, tamarins, Saccharomyces cerevisiae, and Escherichia coli. In addition, sequences from these clones were not detected in plasma or liver tissue of tamarins prior to their inoculation with the GB agent. These sequences were detected by reverse transcription-PCR in acute-phase plasma of tamarins inoculated with the GB agent. Probes derived from two of the seven clones detected an RNA species of > or = 8.3 kb in the liver of a GB-agent-infected tamarin by Northern blot hybridization. Sequence analysis indicated that five of the seven clones encode polypeptides that possess limited amino acid identity with the nonstructural proteins of hepatitis C virus. Extension of the sequences found in the seven clones revealed that plasma from an infected tamarin contained two RNA molecules > 9 kb long. Limited sequence identity with various isolates of hepatitis C virus and the relative positions of putative RNA helicases and RNA-dependent RNA polymerases in the predicted protein products of these molecules suggested that the GB agent contains two unique flavivirus-like genomes.
Asunto(s)
Flavivirus/genética , Flavivirus/aislamiento & purificación , Genoma Viral , Hepatitis Viral Animal/virología , Hepatitis Viral Humana/virología , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Northern Blotting , Clonación Molecular , ADN Viral/sangre , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Saguinus , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Pase SeriadoRESUMEN
Recombinant antigens from hepatitis E virus (HEV) open-reading frames 2 and 3 were expressed in Escherichia coli as cytidine monophosphate-2-keto-3-deoxyoctulosonic acid synthetase (CKS) fusion proteins, purified, and used to develop an EIA for the detection of antibodies. Serologic results were compared with those of previous assays by testing 102 samples from an HEV outbreak in Somalia. This CKS/HEV EIA detected anti-HEV in all 97 sera found reactive previously and in an additional 2 samples, which were shown to be true HEV-positive samples by supplemental peptide and Western blot tests. The CKS/HEV EIA and supplemental assays were then used to determine seroprevalence of HEV worldwide. HEV seroprevalence ranged from 1% to 25%, with higher rates found in Middle Eastern countries. Also, 7%-14% of acute cases of non-A, -B, or -C hepatitis were HEV-positive. Thus, this CKS/HEV EIA appears useful for detecting anti-HEV in various populations.
Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Hepatitis E/inmunología , Hepatitis E/epidemiología , Proteínas Recombinantes de Fusión , Proteínas Virales , África/epidemiología , Donantes de Sangre , Western Blotting , Brotes de Enfermedades , Europa (Continente)/epidemiología , Virus de la Hepatitis E/genética , Humanos , Sueros Inmunes/inmunología , Técnicas para Inmunoenzimas , Japón/epidemiología , Medio Oriente/epidemiología , Sistemas de Lectura Abierta , Prevalencia , Proteínas Recombinantes de Fusión/inmunología , Sensibilidad y Especificidad , Tailandia/epidemiología , Proteínas Virales/síntesis química , Proteínas Virales/inmunologíaRESUMEN
The gene encoding the 12-kDa cytosolic form of human FK506-binding protein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and expressed in three different non-fusion systems in Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the trc promoter vector, pKK233-2, yielded low levels of protein. A second system, which utilized a modified lac promoter and a stronger ribosome-binding site, showed greatly improved expression. A third system, utilizing translational coupling to an upstream segment of kdsB under the control of this modified lac promoter, produced re-hFKPB-12 at a very high level. The re-hFKBP-12 produced via translational coupling was soluble and was shown to have the authentic N terminus. The level of active re-hFKBP-12 produced from this vector was estimated to be 50% of total soluble protein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Biosíntesis de Proteínas , Tacrolimus , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes , Humanos , Operón Lac , Datos de Secuencia Molecular , Nucleotidiltransferasas/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Análisis de Secuencia de ADN , Tacrolimus/análogos & derivados , Proteínas de Unión a TacrolimusRESUMEN
Selected regions of the Borrelia burgdorferi flagellin gene (fla) that exhibit high or low homology with related genes from other bacterial species were amplified by the polymerase chain reaction and expressed as fusion proteins in Escherichia coli. Purified fusion proteins were assayed for antibody reactivity in a microtiter plate enzyme-linked immunosorbent assay with sera from Lyme disease patients as well as syphilitic and normal sera. Immunoglobulin G antibody from Lyme disease patient sera reacted predominantly with the central portion of the protein. The region of the flagellin protein encompassing amino acids 64 to 311 detected nearly all of the immunoglobulin G-positive Lyme sera and only reacted with 1 of 26 syphilis patient serum samples. In contrast, 12 of 26 syphilis patient serum samples and 2 of 47 normal serum samples reacted with the amino terminus of the flagellin protein, whereas 4 of 26 syphilis patient serum samples and 7 of 47 normal serum samples reacted with the carboxyl terminus. The central region containing amino acids 64 to 311 may be employed diagnostically to differentiate antibodies to B. burgdorferi from antibodies to Treponema pallidum. In addition, this region also was recognized by immunoglobulin M in the Lyme patient sera, indicating its potential usefulness as a marker for early Lyme disease.
Asunto(s)
Grupo Borrelia Burgdorferi/inmunología , Flagelina/inmunología , Enfermedad de Lyme/diagnóstico , Sífilis/diagnóstico , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Secuencia de Bases , Clonación Molecular , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Flagelina/genética , Humanos , Inmunoglobulina M/análisis , Datos de Secuencia Molecular , Nucleotidiltransferasas/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L in Escherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a approximately 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed in E. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.