RESUMO
A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) method was developed for simultaneous detection and typing/subtyping of influenza viruses A/H1, A/H3 or B, and respiratory syncytial viruses A or B, followed by DNA semiquantitation using the Agilent 2100 Bioanalyzer. Such method provides a rapid, specific and sensitive diagnostic tool for detection and semiquantification of respiratory illness specimens.
Assuntos
Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Vírus Sinciciais Respiratórios/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Pré-Escolar , Humanos , Lactente , Sensibilidade e EspecificidadeRESUMO
Hemagglutinin sequences of 146 human influenza A/H3N2 strains identified in respiratory specimens from Asia and Europe during the 2001-2003 influenza seasons were analyzed by DNA sequencing. Our results suggest that four amino acid substitutions, L25I, H75Q, H155T, and Q156H, led to the antigenic conversion of the previously predominant A/Panama/2007/99-like strains to the more recent A/Fujian/411/2002-like strains.
Assuntos
Substituição de Aminoácidos , Evolução Molecular , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/epidemiologia , Ásia/epidemiologia , DNA Viral/análise , Europa (Continente)/epidemiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Influenza Humana/virologia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
One-hundred five influenza B-positive specimens obtained from southeast Asia in 2002 were categorized on the basis of DNA sequencing of HA1 gene as well as real-time PCR analysis of the NA gene. Phylogenetic analysis of the HA1 gene sequences showed that the majority of the viruses (96.2%) belonged to the B/Victoria/2/87 lineage, while a smaller percentage of the viruses (3.8%) belonged to the B/Yamagata/16/88 lineage. The B/Yamagata/16/88 viruses displayed significant antigenic drift in the deduced amino acid sequences of the HA1 protein, and the B/Victoria/2/87-like viruses consisted of B/Hong Kong/1351/02-like (72.3%) and B/Hong Kong/330/01-like (27.7%) viruses. The B/Hong Kong/1351/02-like viruses were reassortants with the HA gene belonging to the B/Victoria/2/87 lineage and the NA gene belonging to the B/Yamagata/16/88 lineage, whereas both the HA and NA genes of B/Hong Kong/330/01 virus belonged to the B/Victoria/2/87 lineage. In this study, however, all the B/Hong Kong/330/01-like isolates exhibited the B/Yamagata/16/88-like NA gene, which likely resulted from reassortment of B/Hong Kong/330/01 and B/Hong Kong/1351/02 viruses during coinfection. Additional molecular characterization of the six internal genes showed that the M, NS, PA, and PB2 genes of the new variants were B/Hong Kong/1351/02 in origin, whereas the NP and PA genes retained the B/Hong Kong/330/01 origin. Interestingly, these new variants all appeared late in the year 2002. These results support the notion that influenza B viruses continued to evolve through antigenic drift and shift.
Assuntos
Variação Genética , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Influenza Humana/virologia , Sequência de Bases , Pré-Escolar , Primers do DNA , Humanos , Lactente , Vírus da Influenza B/classificação , Influenza Humana/diagnóstico , Influenza Humana/epidemiologia , Dados de Sequência Molecular , New York/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Forty-nine influenza B virus isolates collected in Belgium, Finland, Spain, and Israel during the 2001-2002 winter season were categorized into either of two lineages, B/Yamagata/16/88 or B/Victoria/2/87, based on the phylogenetic studies of HA1 sequences. The data trace the geographic spread of B/Victoria/2/87-like viruses and support the emergence of B/Hong Kong/1351/02-like viruses, possibly due to selective advantages of reassortment.
Assuntos
Vírus da Influenza B/classificação , Influenza Humana/epidemiologia , Filogenia , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Europa (Continente)/epidemiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza B/genética , Israel/epidemiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
To investigate the role of Reg Ialpha in human inflammatory bowel disease (IBD), we made two phage-displayed single chain variable fragment (scFv) libraries from rabbits immunized with recombinant or native human Reg Ialpha. After one to three rounds of panning, we were able to isolate phage-displaying scFvs, which bound to human Reg Ialpha. Anti-Reg Ialpha scFvs from both libraries showed similar immunoreactivity to different processed forms of the protein. Despite several DNA fingerprint patterns among these clones, their deduced amino acid sequences are highly homologous with 100% identity in the complementarity-determining regions (CDRs) of the variable segment of heavy chain (VH) region and a small variation in the CDR1 of the variable segment of light chain (VL) region. We also expressed and purified soluble myc-tagged or glutathione S-transferase (GST) fusion scFv proteins from bacteria. Immunohistochemical studies using one of our anti-Reg Ialpha scFv antibodies showed prominent staining in the metaplastic Paneth cell population and light staining in the lamina propria. This scFv antibody is now being used for studies of the role of Reg Ialpha in human IBD.
