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1.
Influenza Other Respir Viruses ; 14(2): 204-209, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31651085

RESUMO

BACKGROUND: Cell-based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation-associated antigenic changes observed in egg-based vaccines. Seed viruses for cell-based vaccines can be prepared from clinical specimens by cell culture; however, clinical samples risk harboring respiratory viruses other than influenza virus. Therefore, it is necessary to investigate the patterns of co-infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses. METHODS: A total of 341 clinical specimens were collected from patients with influenza or influenza-like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses. The patterns of co-infection were statistically analyzed with Fisher's exact test. The samples with double or triple infections were passaged in suspension MDCK cells (MDCK-S), adherent MDCK cells (MDCK-A), and LLC-MK2D cells. Cell-passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses. RESULTS: Double infections were detected in 8.5% and triple infections in 0.9% of the collected clinical specimens. We identified four pairs of viruses with significant correlation. For all samples with double and triple infection, MDCK-S and MDCK-A could selectively propagate influenza viruses, while eliminating all contaminating viruses. In contrast, LLC-MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK-S and MDCK-A. CONCLUSIONS: Both MDCK-S and MDCK-A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg-adaptation mutations.


Assuntos
Células Madin Darby de Rim Canino/virologia , Orthomyxoviridae/isolamento & purificação , Cultura de Vírus/métodos , Animais , Linhagem Celular , Cães , Humanos , Orthomyxoviridae/crescimento & desenvolvimento , Vacinas Virais
2.
Vaccine ; 37(43): 6526-6534, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31500967

RESUMO

Suspension Madin-Darby canine kidney (MDCK) cells (MDCK-N), adherent MDCK cells (MDCK-C), and adherent rhesus monkey kidney LLC-MK2 cells (LLC-MK2D) were systematically evaluated for the preparation of influenza vaccine seed viruses for humans on the basis of primary virus isolation efficiency, growth ability, genetic stability of the hemagglutinin (HA) and neuraminidase (NA) genes, and antigenic properties in hemagglutination inhibition (HI) test of each virus isolate upon further passages. All the subtypes/lineages of influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), B-Victoria, and B-Yamagata) were successfully isolated from clinical specimens by using MDCK-N and MDCK-C, whereas LLC-MK2D did not support virus replication well. Serial passages of A(H1N1) viruses in MDCK-N and MDCK-C induced genetic mutations of HA that resulted in moderate antigenic changes in the HI test. All A(H1N1)pdm09 isolates from MDCK-C acquired amino acid substitutions at the site from K153 to N156 of the HA protein, which resulted in striking antigenic alteration. In contrast, only 30% of MDCK-N isolates showed amino acid changes at this site. The frequency of MDCK-N isolates with less than two-fold reduction in the HI titer was as high as 70%. A(H3N2) and B-Yamagata isolates showed high antigenic stability and no specific amino acid substitution during passages in MDCK-N and MDCK-C. B-Victoria isolates from MDCK-N and MDCK-C acquired genetic changes at HA glycosylation sites that greatly affected their antigenicity. When these cell isolates were applied to passages in hen eggs, A(H1N1), B-Victoria, and B-Yamagata viruses grew well in eggs, while none of the cell isolates of A(H3N2) viruses did. Thus, we demonstrate that MDCK-N might be useful for the preparation of influenza vaccine seed viruses.


Assuntos
Vírus da Influenza A/classificação , Vírus da Influenza A/fisiologia , Cultura de Vírus/métodos , Replicação Viral , Animais , Linhagem Celular , Cães , Hemaglutininas Virais/genética , Vacinas contra Influenza , Rim/citologia , Rim/virologia , Macaca mulatta , Células Madin Darby de Rim Canino , Mutação , Neuraminidase/genética , RNA Viral/genética
3.
J Med Microbiol ; 62(Pt 4): 610-617, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23329324

RESUMO

Detailed genetic analysis was carried out of the VP4/VP2 coding region in human rhinovirus species C (HRV-C) strains detected in patients with acute respiratory infection in Japan. Phylogenetic trees were constructed by the neighbour-joining (NJ) and maximum-likelihood (ML) methods. The NJ phylogenetic tree assigned 11 genotypes to the present strains, whilst the ML tree showed that the strains diversified sometime in the early 1870 s. Moreover, the pairwise distance among the present strains was relatively long, and the rate of molecular evolution of the coding region was rapid (3.07 × 10(-3) substitutions per site per year). The results suggest that the present HRV-C strains have a wide genetic divergence and a unique evolutionary timescale.


Assuntos
Variação Genética , Infecções por Picornaviridae/virologia , Infecções Respiratórias/virologia , Rhinovirus/classificação , Rhinovirus/genética , Proteínas Virais/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Análise por Conglomerados , Evolução Molecular , Feminino , Genótipo , Humanos , Lactente , Japão , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Rhinovirus/isolamento & purificação , Análise de Sequência de DNA , Adulto Jovem
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