RESUMO
Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, Zika virus/Homo sapiens-tc/THA/2014/SV0127-14 and Zika virus/H. sapiens-tc/PHL/2012/CPC-0740, isolated from the blood of patients collected in Thailand, 2014, and the Philippines, 2012, respectively. Sequencing and phylogenetic analysis showed that both strains belong to the Asian lineage.
RESUMO
BACKGROUND: Influenza virus (IFV) isolates obtained from mammalian cell cultures are valuable reagents used for vaccine production, antigenic characterization, laboratory assays, and epidemiological and evolutionary studies. Complete genomic comparison of IFV isolates with their original clinical specimens provides insight into cell culture-driven genomic changes which may sequentially alter the virus phenotype. OBJECTIVES: The genome of the viral isolates and of the viruses in the clinical specimens was examined by deep sequencing in order to determine nucleotide heterogeneity (measured number of variances or numbers of mixed bases) as a marker for IFV population diversity. STUDY DESIGN: Clinical respiratory specimens were collected between July and October 2012 and identified by RT-PCR as positive for influenza A H3N2 or H1N1, or influenza B. The viruses in the clinical specimens were amplified using mammalian cell culture. Next generation sequencing (NGS) was used to investigate genomic differences between IFV isolates and their corresponding clinical specimens. RESULTS: There was less nucleotide heterogeneity in 5 of 6 viral isolates compared to the corresponding clinical specimens, especially for influenza B. A phylogenetic analysis of the hemagglutinin (HA) gene consensus sequences obtained from deep and Sanger sequencing showed that the viral isolates and their corresponding clinical specimens contained the same IFV strains with less than 5% pair-wise genetic distance. CONCLUSION: The IFV sequence data analysis detected a substantial decrease in nucleotide heterogeneity from clinical specimens to viral cultures in 5 out of 6 investigated cases.
Assuntos
Variação Genética , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza B/classificação , Influenza Humana/virologia , Adolescente , Adulto , Criança , Pré-Escolar , Monitoramento Epidemiológico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Masculino , Dados de Sequência Molecular , RNA Viral/genética , Análise de Sequência de DNA , Adulto JovemRESUMO
The prevailing global spread of four dengue virus (DENV) serotypes and the resultant co-circulation of multiple serotypes in the same region have invariably led to conditions supporting the periodic occurrence of simultaneous infection of individuals with more than one DENV serotype. This raises the issue of how best to detect concurrent multiple infections. We report here the use of a nested reverse transcription-polymerase chain reaction (RT-PCR) assay, which detected concurrent infection with three DENV serotypes (DENV-1/DENV-2/DENV-3) and two serotypes (DENV-1/DENV-2 and DENV-2/DENV-4), respectively, in three serum specimens from Thai children hospitalized during the dengue epidemic of 2000-2001. In contrast, an enzyme-linked immunosorbent assay used previously for virus serotype identification failed to detect multiple DENV serotypes in these specimens. Serotype identification by RT-PCR was confirmed by sequence analysis of each amplified PCR product. Phylogenetic analyses performed on PCR-amplified DNA fragments further supported the occurrence of concurrent infections with multiple DENV serotypes in these children. Although the sample set was small, our data suggest that nested RT-PCR is an effective method for the detection of concurrent DENV infections.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/epidemiologia , Dengue/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sorotipagem/métodos , Adolescente , Criança , Pré-Escolar , Vírus da Dengue/classificação , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Filogenia , Estudos Retrospectivos , Tailândia/epidemiologiaRESUMO
Monoclonal antibodies were produced against two distinct Thai dengue-2 (DEN-2) virus strains isolated in 1980 from dengue haemorrhagic fever patients. Nine of 36 hybridomas produced monoclonal IgG antibodies which reacted in radioimmune precipitation assays with the NS1 non-structural protein (42,000 mol. wt.) from DEN-2-infected C6/36 (Aedes albopictus) cells. The virus specificity of NS1-reactive monoclonal antibodies was determined by indirect immunofluorescence assays using LLC-MK2 cells infected with either the Thai 1980 DEN-2 isolates, prototype DEN viruses (four serotypes), Japanese encephalitis (JE), Murray Valley encephalitis, West Nile, Wesselbron or Tembusu viruses. Eight of the monoclonal antibody preparations were DEN-2-serotype specific. One preparation defined a special serological relationship between DEN-2 and JE viruses. Four preparations had detectable complement fixation titres using Thai DEN-2 virus antigen. Six spatially unique epitopes were identified using competitive binding assays.