RESUMO
Borna disease virus (BDV) is a neurotropic, as yet unclassified, non-segmented, negative-sense, single-strand RNA virus. Natural infection with this virus has been reported to occur in horses and sheep. In addition, antibodies to BDV in plasma or BDV RNA in peripheral blood mononuclear cells (PBMCs) were also found in patients with neuropsychiatric diseases. We describe here the possible link between the patients with chronic fatigue syndrome (CFS) and infection with BDV.
Assuntos
Anticorpos Antivirais/líquido cefalorraquidiano , Antígenos Virais/imunologia , Doença de Borna/virologia , Vírus da Doença de Borna/isolamento & purificação , Síndrome de Fadiga Crônica/virologia , Proteínas Virais/imunologia , Adulto , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Doença de Borna/imunologia , Vírus da Doença de Borna/genética , Vírus da Doença de Borna/imunologia , Síndrome de Fadiga Crônica/líquido cefalorraquidiano , Síndrome de Fadiga Crônica/imunologia , Humanos , Japão , RNA Viral/líquido cefalorraquidiano , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais/genéticaRESUMO
A protocol was developed for a highly sensitive detection of viral RNA in blood specimens by reverse transcription coupled with a nested polymerase chain reaction. Using Japanese encephalitis virus (JEV) as a model, the optimized reverse transcription-polymerase chain reaction (ORTPCR) detects as few as 3-5 virions in 0.1 ml of whole blood specimens. The effectiveness of this assay system is confirmed by diagnosis of human hepatitis C viral (HCV) infection.
Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Viral/sangue , Animais , Sequência de Bases , Criança , Vírus da Encefalite Japonesa (Subgrupo)/isolamento & purificação , Hepacivirus/isolamento & purificação , Hepatite C/microbiologia , Humanos , Camundongos , Dados de Sequência Molecular , DNA Polimerase Dirigida por RNA , Sensibilidade e EspecificidadeRESUMO
Hepatitis C virus (HCV) is a positive strand RNA virus with certain similarity to flaviviruses and pestiviruses. To examine the processing and possible assembly of HCV proteins, we constructed a recombinant vaccinia virus that expresses a full-length genomic RNA, infected chimp liver cells with the virus, and analyzed HCV-related protein products by immunofluorescent antibody staining and Western blot detection with mouse monoclonal antibodies. The putative core, envelope, and NS1 and NS3 proteins that yielded from this recombinant were 22, 32, 53 to 58, and 65 kDa in size, respectively. The NS4 protein was unexpectedly small, with an estimated molecular weight of 7 kDa, and the NS5 protein was found to be further cleaved into 52-kDa NS5a and 58-kDa NS5b proteins, the latter of which contains a hallmark of RNA replicase. A point mutation in the putative protease domain of NS3 resulted in a failure in the production of NS3, NS4, NS5a, and NS5b, but coexpression of NS3 restored the proper processing of these proteins, demonstrating that NS3, the putative viral protease, is essential for the production of these nonstructural proteins. Thus, HCV strikingly resembles pestiviruses in the size and the processing mode of the nonstructural proteins, particularly NS4 and NS5.
Assuntos
Endopeptidases/metabolismo , Genes Virais/genética , Hepacivirus/enzimologia , Hepacivirus/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/metabolismo , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Sequência de Bases , Células Cultivadas , DNA Helicases/genética , DNA Helicases/metabolismo , Endopeptidases/genética , Escherichia coli/genética , Genoma Viral , Hepacivirus/genética , Humanos , Fígado/citologia , Dados de Sequência Molecular , Pan troglodytes , Mutação Puntual , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Transfecção , Vaccinia virus/genética , Proteínas não Estruturais Virais/genéticaRESUMO
The localization of hepatitis C virus-infected hepatocytes in the human liver remains unclear despite the development of a serological assay for the antibody to hepatitis C virus. We studied their localization immunohistochemically with monoclonal antibodies to core, envelope and NS3 antigens of hepatitis C virus. We examined 48 liver biopsy samples from C100-3 antibody-positive patients with chronic liver disease (chronic persistent hepatitis, 5 cases; chronic active hepatitis, 41 cases; cirrhosis, 2 cases) and 12 liver biopsy samples from C100-3 antibody-negative patients with chronic liver disease (type B chronic hepatitis, 8 cases; alcoholic liver disease, 4 cases). In the C100-3 antibody-positive group, positive immunostaining for core antigen, envelope antigen and NS3 antigen was found in 23% (11 of 48), 24% (11 of 45) and 24% (11 of 46), respectively. Negative results were obtained in the C100-3 antibody-negative group. Hepatocytes with positive staining were scattered in the lobules, and they were found in the same regions irrespective of whether the antibody to core antigen, to envelope antigen or to NS3 antigen was used. Each positive cell was strongly stained in the cytoplasm; these decorations disappeared after absorption of the primary antibody with purified antigen. mean ALT levels in the patients with positive immunostaining for core, envelope or NS3 antigen (174.8 +/- 105.7 U/L) tended to be higher than in those with negative immunostaining (142.0 +/- 93.8 U/L). On histological evaluation of liver specimens with a scoring system of the histological activity index, intralobular inflammation and fibrosis had higher scores for samples with positive rather than negative immunostaining (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos Virais/análise , Capsídeo/análise , Hepacivirus/isolamento & purificação , Hepatite C/microbiologia , Fígado/microbiologia , Proteínas do Core Viral/análise , Proteínas do Envelope Viral/análise , Adolescente , Adulto , Idoso , Capsídeo/imunologia , Doença Crônica , Feminino , Genoma Viral , Hepacivirus/genética , Hepacivirus/imunologia , Antígenos da Hepatite C , Humanos , Imuno-Histoquímica , Interferon-alfa/farmacologia , Fígado/química , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Proteínas do Core Viral/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais ViraisRESUMO
Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis, which often develops into malignant chronic diseases, including liver cirrhosis and hepatocellular carcinoma. We have cloned from human carriers overlapping cDNAs (9,416 bp) covering the entire coding region of the HCV genome. The latter encodes a 3,010-amino-acid polyprotein. In addition, there are 332 and 54 bases of 5' and 3' noncoding sequences, respectively. Our HCV strain has a 77% nucleic acid identity to the HCV strain cloned by workers at Chiron Corporation. The hydrophobicity profile of the putative polyprotein is similar to those of flaviviruses, but it has limited amino acid homology to polyproteins of flaviviruses and other viruses, indicating that HCV is at most distantly related to flaviviruses.
Assuntos
DNA Viral/genética , Genes Virais , Hepacivirus/isolamento & purificação , RNA Viral/sangue , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Portador Sadio , Clonagem Molecular , Hepacivirus/genética , Humanos , Dados de Sequência Molecular , Conformação Proteica , RNA Viral/genética , RNA Viral/isolamento & purificação , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Proteínas Virais/genéticaRESUMO
Complementary DNAs to the 5' proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5' terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5'-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.
Assuntos
Vírus da Dengue/genética , Genes Virais , Genes , RNA Viral , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA , Vírus da Dengue/classificação , Dados de Sequência Molecular , Filogenia , Plasmídeos , RNA Viral/genética , Mapeamento por Restrição , Proteínas Virais/genética , Proteínas Estruturais Virais , Cultura de VírusRESUMO
The complete nucleotide sequence of the Japanese encephalitis virus (JEV) genome RNA was determined. The JEV genome contains 10,976 nucleotides and encodes a single long open reading frame (ORF) of 10,296 nucleotides corresponding to 3432 amino acid residues. This long polypeptide is thought to be cleaved into three structural proteins and several nonstructural proteins of the virus. The genetic location of the three structural proteins was determined by comparing the deduced amino acid sequence from the nucleotide sequence with the N-terminal amino acid sequences that were determined from the three purified structural proteins. The C-terminal region of the ORF may encode a RNA-dependent RNA polymerase which has significant sequence homology with those of other RNA viruses.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Genes Virais , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Códon , DNA/genética , Genes , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Precursores de Proteínas/genética , Homologia de Sequência do Ácido NucleicoRESUMO
A safe, effective and economical vaccine is required for the prevention of Japanese encephalitis (JE), a disease with high mortality and grave sequelae, which is prevalent in Japan and other countries in east, south-east and southern Asia. As the initial step to produce a second-generation vaccine, recombinant DNA technology was utilized to express the JE virus envelope glycoprotein V3 (E) gene in yeast cells.This report describes the construction of a yeast expression vector in which a cDNA clone covering the V3 gene was connected to the acid-phosphatase promoter of a yeast vector plasmid. Successful expression of the V3 gene was detected by ELISA and Western blotting using monoclonal antibodies against JE V3. Immunization of mice with the V3 antigen expressed in yeast produced limited but definite levels of anti-JE antibodies which could neutralize JE virus. The results are an encouraging step in the development of a practical second-generation JE vaccine.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/prevenção & controle , Genes Virais , Glicoproteínas/genética , Proteínas do Envelope Viral/genética , Vacinas Virais , DNA Recombinante , Humanos , LevedurasRESUMO
The 5' region of the Japanese encephalitis virus (JEV) RNA was cloned and 3000 nucleotides (nt) were determined by sequencing DNA complementary to viral RNA, and genomic RNA, using oligodeoxynucleotide primers and the dideoxy chain-termination reaction. Comparison of the nt sequence and the reduced amino-acid sequence of JEV with those of other flaviviruses showed significant homologies, which allowed locations to be assigned for three structural proteins.
