RESUMO
We report on the occurrence and diversity of noroviruses in children (younger than 5 years old of age) from a low-income urban area in Rio de Janeiro, Brazil. Sixty-one stool specimens collected from children between 1 and 4 years old with acute diarrhoeic episodes (ADE) and non-ADE were investigated. RT-qPCR and sequencing of PCR products after conventional RT-PCR analysis were performed. Noroviruses were detected in 29 (47.5%) samples: 21 (46.7%) from cases with ADE and 8 (50%) from non-ADE cases. Molecular characterization showed 10 different genotypes circulating in this community between November 2014 and April 2018.
Assuntos
Gastroenterite/virologia , Variação Genética/genética , Norovirus/genética , Brasil , Pré-Escolar , Fezes/virologia , Gastroenterite/diagnóstico , Genótipo , Humanos , Lactente , Norovirus/isolamento & purificação , Filogenia , Pobreza , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
The Histo-blood group antigens (HBGA) are host genetic factors associated with susceptibility to rotavirus (RV) and human norovirus (HuNoV), the major etiological agents of viral acute gastroenteritis (AGE) worldwide. The FUT2 gene expressing the alpha-1, 2-L- fucosyltransferase enzyme is important for gut HBGA expression, and also provides a composition of the phenotypic profile achieved through mutations occurring in populations with different evolutionary histories; as such, it can be considered a genetic population marker. In this study, Lewis and secretor HBGA phenotyping was performed using 352 saliva samples collected from children between three months and five years old born in the Amazon (Brazil, Venezuela and English Guyana) presenting AGE or acute respiratory infection (ARI), the latter considered as control samples. The total of children phenotyped as secretors was 323, corresponding to 91.80%. From these, 207 (58.80%) had a Le (aâ¯+â¯b+) profile. The HBGA profiles were equally found in children with AGE as well as with ARI. The rs1047781 of the FUT2 gene was not detected in DNA from saliva cells with a Le (a+b+) profile. However, mutations not yet described in the FUT2 gene were observed: missense 325A>T, 501C>T, 585C>T, 855A>T and missense substitutions 327C>T [S (Ser) > C (Cys)], 446 T>C [L(Leu) > P(Pro)], 723C>A [N(Asn) > K(Lys)], 724A>T [I(Ile) > F(Phe)], 736C>A [H(His) > N(Asn)]. The SNP distribution in the FUT2 gene of the analyzed samples was very similar to that described in Asian populations, including indigenous tribes.
Assuntos
Infecções por Caliciviridae/epidemiologia , Fucosiltransferases/genética , Gastroenterite/epidemiologia , Predisposição Genética para Doença/etnologia , Antígenos do Grupo Sanguíneo de Lewis/genética , Infecções por Rotavirus/epidemiologia , Doença Aguda/epidemiologia , Brasil , Infecções por Caliciviridae/etnologia , Pré-Escolar , Feminino , Fucosiltransferases/sangue , Gastroenterite/virologia , Marcadores Genéticos , Humanos , Lactente , Antígenos do Grupo Sanguíneo de Lewis/sangue , Masculino , Polimorfismo de Nucleotídeo Único , Infecções Respiratórias , Infecções por Rotavirus/etnologia , Saliva/virologia , Venezuela , Galactosídeo 2-alfa-L-FucosiltransferaseAssuntos
Humanos , RNA Viral/genética , Vírus da Hepatite B/genética , Técnicas de Diagnóstico Molecular/métodos , Farmacorresistência Viral/genética , Mutação/genética , Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/genética , Lamivudina/farmacologiaAssuntos
Farmacorresistência Viral/genética , Vírus da Hepatite B/genética , Técnicas de Diagnóstico Molecular/métodos , Mutação/genética , RNA Viral/genética , Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Lamivudina/farmacologia , DNA Polimerase Dirigida por RNA/genéticaRESUMO
The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.
Assuntos
Antígenos Virais/biossíntese , Antígenos Virais/isolamento & purificação , Biotecnologia/métodos , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/isolamento & purificação , Expressão Gênica , Animais , Antígenos Virais/genética , Baculoviridae/genética , Proteínas do Capsídeo/genética , Linhagem Celular , Vetores Genéticos , Células HEK293 , Humanos , Insetos , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Rotavirus/genéticaRESUMO
The hepatitis B virus surface protein (HBsAg) displays the major B cells antigenic determinants that can induce protective immunity and prevent the hepatitis B virus (HBV) infection, a major health problem. A panel of murine monoclonal antibodies against the HBsAg (MAb anti-HBs), raised after mice immunization with a pool of plasma of hepatitis chronic carriers, has been established. Mainly using simple immunological tools such as enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, we could trace the location of the epitopes on the HBsAg determinants. We also report the use of two specific methodology approaches based on molecular biology and biochemical techniques such as, respectively, cloning and expression of preS1 major neutralizing epitope of the HBsAg in Escherichia coli and ELISA accomplished to chemical reduction with dithiothreitol (DTT), which were able to complete the MAb anti-HBs characterization. Our results showed that the majority of the MAbs anti-HBs were directed to the HBV common determinant a. One MAb recognizes a discontinuous epitope present in all forms of the HBsAg when evaluated by Western blot.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Alquilação , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Far-Western Blotting , Western Blotting , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/imunologia , Antígenos de Superfície da Hepatite B/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
Different mammalian cells have been used successfully for the expression of the hepatitis B surface antigen (HBsAg). The patterns of expression of the HBsAg seem to depend on the cell type and the number of gene copies integrated into cellular genome. The expression of an HBsAg fused to histidine tag (His-HBsAg), had not been reported in mammalian cells. This paper describes a semi-quantitative polymerase chain reaction (PCR) employed to investigate the patterns of expression of His-HBsAg in stably transfected Chinese hamster ovary (CHO) cell clones. pcDNA4CR20, a mammalian expressing vector, encoding His-HBsAg, was constructed. The correlation between His-HBsAg expression and the number of integrated copies of the HBsAg gene into cell clones genome was evaluated by the semi-quantitative PCR, with limit dilutions of the genomic DNA as template. The results show a positive correlation between the expression levels of His-HBsAg with the number of the HBsAg gene copies integrated into CHO cell clones. The approach of a semi-quantitative PCR proved to be a good and non-expensive alternative strategy to analyze the patterns of expression of integrated genes into CHO cells genome.
