RESUMO
The Hepatitis A virus (HAV) has been classified in seven different genotypes, which include human (I, II, III, and VII) and simian (IV, V, and VI) groups. The sequence analysis of HAV strains contributes to the molecular epidemiology of the virus. Although the infection with HAV is endemic in Argentina and vaccination is being implemented in this country, using both IA and IB strains, there are very few data on the genotypes of the circulating viruses. On the basis of the sequences of 20 isolates collected in Buenos Aires during a 2-year period (extended to 3 years by two additional specimens), we observed the presence of a single sub-genotype, IA, but with a high genetic diversity. We analyzed the VP1-2A junction and also the VP3-VP1 region. Most of the Argentine isolates grouped in at least two clusters. One of these was related to South American strains, thus suggesting a co-circulation of related isolates in neighbor countries. The other cluster was composed only of Argentine specimens. Other sequences were more scattered along the phylogenetic tree. However, we demonstrated that a consistent genetic relatedness of sequences could only be inferred on the basis of a more extensive sequencing of each isolate.
Assuntos
Vírus da Hepatite A/genética , Hepatite A/virologia , Adulto , Sequência de Aminoácidos , Argentina/epidemiologia , Capsídeo/genética , Variação Genética , Genótipo , Hepatite A/epidemiologia , Vírus da Hepatite A/classificação , Vírus da Hepatite A/isolamento & purificação , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Proteínas Estruturais Virais/genéticaRESUMO
Astroviruses require the proteolytic cleavage of the capsid protein to infect the host cell. Here we describe the processing pathway of the primary translation product of the structural polyprotein (ORF2) encoded by a human astrovirus serotype 8 (strain Yuc8). The primary translation product of ORF2 is of approximately 90 kDa, which is subsequently cleaved to yield a 70-kDa protein (VP70) which is assembled into the viral particles. Limited trypsin treatment of purified particles containing VP70 results in the generation of polypeptides VP41 and VP28, which are then further processed to proteins of 38.5, 35, and 34 kDa and 27, 26, and 25 kDa, respectively. VP34, VP27 and VP25 are the predominant proteins in fully cleaved virions, which correlate with the highest level of infectivity. Processing of the VP41 protein to yield VP38.5 to VP34 polypeptides occurred at its carboxy terminus, as suggested by immunoblot analysis using hyperimmune sera to different regions of the ORF2, while processing of VP28 to generate VP27 and VP25 occurred at its carboxy and amino terminus, respectively, as determined by immunoblot, as well as by N-terminal sequencing of those products. Based on these data, the processing pathway for the 90-kDa primary product of astrovirus Yuc8 ORF2 is presented.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Glicoproteínas/metabolismo , Mamastrovirus/metabolismo , Sequência de Aminoácidos , Células CACO-2 , Capsídeo/genética , Glicoproteínas/genética , Humanos , Mamastrovirus/classificação , Mamastrovirus/genética , Mamastrovirus/patogenicidade , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sorotipagem , Tripsina , VirulênciaRESUMO
A potyvirus was found causing yellow mosaic and veinal banding in sweetpepper in Central and Southeast Brazil. The sequence analysis of the 3' terminal region of the viral RNA revealed a coat protein of 278 amino acids, followed by 275 nucleotides in the 3'-untranslated region preceding a polyadenylated tail. The virus shared 77.4% coat protein amino acid identity with Pepper severe mosaic virus, the closest Potyvirus species. The 3'-untranslated region was highly divergent from other potyviruses. Based on these results, the virus found in sweetpepper plants could be considered as a new potyvirus. The name Pepper yellow mosaic virus (PepYMV) is suggested.
Assuntos
Capsicum/virologia , Genoma Viral , Doenças das Plantas/virologia , Potyvirus/classificação , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Brasil , Capsídeo/genética , Clonagem Molecular , Dados de Sequência Molecular , Filogenia , Potyvirus/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Rotavirus is the most-common cause of severe diarrhea in young children. Complete rotavirus characterization includes determination of the antigenic type of the two outer capsid proteins, VP7 and VP4, designated G and P types, respectively. During a nationwide rotavirus surveillance study, genotype G4 frequency increased during the second year. To evaluate further the mechanism of emergence and the relationship among G4 strains, the genetic diversity of VP7 capsid protein in these samples was studied in detail. Overall nucleotide sequence divergence ranged from less than 0.1 to 19.5%, a higher divergence than that observed for other rotavirus G types (0.1 to 9%). Sequences were classified into two major lineages (designated I and II) based on their nucleotide distances. The most heterogeneous lineage was further subdivided into four sublineages (designated Ia to Id). Most Argentine sequences were of sublineages Ib and Ic, which were confirmed to be independent sequence clusters by parsimony analysis. This study describes different lineages and sublineages within G4 strains and shows that Argentine strains are distantly related to reference strain ST3. The appearance of at least two G4 genotype (sub)lineages during 1998 demonstrates that the increased frequency of these strains was due to the synchronized emergence of different groups of strains.
Assuntos
Antígenos Virais , Proteínas do Capsídeo , Capsídeo/genética , Variação Genética , Infecções por Rotavirus/virologia , Rotavirus/genética , Sequência de Aminoácidos , Argentina/epidemiologia , Capsídeo/química , Pré-Escolar , Genótipo , Humanos , Lactente , Recém-Nascido , Dados de Sequência Molecular , Filogenia , Vigilância da População , Prevalência , Rotavirus/classificação , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/transmissão , Análise de Sequência de DNARESUMO
Echovirus 30 (EV30) is one of the most frequently isolated EVs, causing extensive outbreaks of EV30 aseptic meningitis in temperate climates. EV30 is antigenically heterogeneous, and three major antigenic groups have been defined, although the basis for the antigenic differences is unknown. A reverse transcription-nested PCR which amplifies the 3'-terminal region of the VP1 gene directly from clinical samples was selected for studying EV30 molecular epidemiology, since the major antigenic sites in this region reflect the serotypic pattern of this virus. The different previous approaches to the genetic classification of EV30 were analyzed. A complete study of the EV30 strains was performed by analyzing the sequences from the 112 EV30 strains amplified in this work and the complete set of EV30 strains previously published. A total of 318 strains of EV30 were divided into two broad genotypes (I and II). This classification was supported by the phylogenetic trees obtained from amino acid sequences, and it correlated with the antigenic heterogeneity of the reference strains described in earlier studies. The genotypes could be further divided into subgroups, and these subgroups could be divided into lineages based on their nucleotide distances and levels of bootstrapping. On the other hand, the subgroups and lineages did not result in the same correlation between amino acid and nucleotide differentiation. The molecular epidemiology of EV30 can be compared to influenza virus epidemiology, where prevailing lineages displace the less established lineages on the basis of immune escape. This pattern of evolution is clearly different from that of other enteroviruses. A single lineage at a time appears to be circulating worldwide. This behavior may be related to the epidemic activity of EV30.
Assuntos
Enterovirus Humano B/genética , Infecções por Enterovirus/epidemiologia , Argentina/epidemiologia , Capsídeo/genética , Proteínas do Capsídeo , Enterovirus Humano B/classificação , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , Espanha/epidemiologiaRESUMO
The incidence of human group C rotavirus infections among children and adults in Buenos Aires was evaluated by enzyme linked immunosorbent assays (ELISA) based on recombinant group C VP6 protein (Cowden strain). A total of 976 stool samples taken from patients (ages 6 months to 15 years) with acute diarrhea were tested for the presence of group C rotavirus. Among these, only 10 (1.02%) were group C rotavirus positive by enzyme-linked immunosorbent assay (ELISA) confirmed by absorption with group C VP6 antibodies and by RT-PCR for both VP6 and VP7 genes. The average age (5.86 years) was significantly superior to that in group A-infected patients (1.63 years). Previous exposure to this virus was assessed by detecting specific IgG in sera taken from healthy individuals grouped by age. Of 844 sera tested, 425 (50.3%) were group C IgG positive by ELISA, confirmed by Western blot analysis. The rates of IgG positivity for group A and C rotaviruses during the first years of life indicated that infections with group C are frequent in older children (3-5 years), whereas group A infections are prevalent in infants and young children (6-18 months). This study shows that group C rotavirus infections in Argentine children occur later in life than group A and are relatively common in spite of the low detection rate of this virus.
Assuntos
Proteínas do Capsídeo , Diarreia/epidemiologia , Infecções por Rotavirus/epidemiologia , Adolescente , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Antígenos Virais/análise , Antígenos Virais/imunologia , Argentina/epidemiologia , Western Blotting/métodos , Capsídeo/genética , Capsídeo/imunologia , Criança , Pré-Escolar , Diarreia/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Incidência , Lactente , Prevalência , Rotavirus/genética , Rotavirus/imunologia , Rotavirus/isolamento & purificação , Infecções por Rotavirus/sangue , Infecções por Rotavirus/virologiaRESUMO
The 3' terminal genomic region of a potyvirus causing mosaic disease in several Crotalaria species has been cloned and sequenced. Comparisons of the nucleotide and deduced amino acid (aa) sequences of the cloned cDNA with those from other potyviruses show that the Crotalaria-infecting virus (designated Crotalaria mosaic virus; CrMV) is closely related to Cowpea aphid-borne mosaic virus (CABMV). Maximum identity (95.4%) at the coat protein (CP) aa level was observed between CrMV and a Brazilian strain of CABMV. Phylogenetic analyses derived from the sequence alignments of the CP and 3' untranslated region confirmed the identification of CrMV as a strain of CABMV and the name CABMV-Cr is suggested.
Assuntos
Crotalaria/virologia , Evolução Molecular , Potyvirus/classificação , Potyvirus/genética , Regiões 3' não Traduzidas/genética , Brasil , Capsídeo/genética , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
An outbreak of paralytic poliomyelitis occurred in the Dominican Republic (13 confirmed cases) and Haiti (8 confirmed cases, including 2 fatal cases) during 2000-2001. All but one of the patients were either unvaccinated or incompletely vaccinated children, and cases occurred in communities with very low (7 to 40%) rates of coverage with oral poliovirus vaccine (OPV). The outbreak was associated with the circulation of a derivative of the type 1 OPV strain, probably originating from a single OPV dose given in 1998-1999. The vaccine-derived poliovirus associated with the outbreak had biological properties indistinguishable from those of wild poliovirus.
Assuntos
Surtos de Doenças , Poliomielite/epidemiologia , Poliomielite/virologia , Vacina Antipólio Oral/efeitos adversos , Poliovirus/genética , Poliovirus/patogenicidade , Regiões 5' não Traduzidas , Adolescente , Animais , Capsídeo/genética , Proteínas do Capsídeo , Criança , Pré-Escolar , República Dominicana/epidemiologia , Feminino , Genes Virais , Haiti/epidemiologia , Humanos , Programas de Imunização , Lactente , Masculino , Camundongos , Dados de Sequência Molecular , Mutação Puntual , Poliomielite/prevenção & controle , Poliomielite/transmissão , Poliovirus/classificação , Poliovirus/isolamento & purificação , Vigilância da População , Recombinação Genética , Vacinação , VirulênciaRESUMO
The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.
Assuntos
Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Vírus Norwalk/metabolismo , Replicon , Montagem de Vírus , Animais , Células CACO-2 , Capsídeo/genética , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/fisiologia , Vetores Genéticos , HumanosRESUMO
The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ss-glucuronidase (ssGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135-160 of structural protein VP1 (VP135-160), fused to the ssGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135-160, analyzed by Western blot, were efficiently selected based on their levels of ssGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135-160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.