RESUMO
BACKGROUND: Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses. METHOD: Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air-liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique. RESULTS: Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively). CONCLUSION: We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates.
Assuntos
Coronavirus/crescimento & desenvolvimento , Coronavirus/isolamento & purificação , Células Epiteliais/virologia , Orthomyxoviridae/crescimento & desenvolvimento , Orthomyxoviridae/isolamento & purificação , Mucosa Respiratória/virologia , Cultura de Vírus/métodos , Coronavirus/genética , Efeito Citopatogênico Viral , Células Epiteliais/citologia , Células Epiteliais/imunologia , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/crescimento & desenvolvimento , Vírus da Influenza B/isolamento & purificação , Pessoa de Meia-Idade , Orthomyxoviridae/genética , Orthomyxoviridae/imunologia , Cultura Primária de Células , Mucosa Respiratória/citologia , Replicação ViralRESUMO
Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to insufficient pressure to maintain gene integrity of non-essential genes. We present here the first full-length genome sequence of two clinical isolates. Each encoded gene was compared to Inf-1. In general, little sequence changes were noted, most could be attributed to genetic drift, since the clinical isolates originate from 2009 to 2010 and VR740 from 1962. Hot spots of substitutions were situated in the S1 region of the Spike, the nucleocapsid gene, and the non-structural protein 3 gene, whereas several deletions were detected in the 3'UTR. Most notable was the difference in genome organization: instead of an ORF4A and ORF4B, an intact ORF4 was present in clinical isolates.
