RESUMEN

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.

Asunto(s)

Coronavirus/crecimiento & desarrollo , Coronavirus/aislamiento & purificación , Células Epiteliales/virología , Orthomyxoviridae/crecimiento & desarrollo , Orthomyxoviridae/aislamiento & purificación , Mucosa Respiratoria/virología , Cultivo de Virus/métodos , Coronavirus/genética , Efecto Citopatogénico Viral , Células Epiteliales/citología , Células Epiteliales/inmunología , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza B/crecimiento & desarrollo , Virus de la Influenza B/aislamiento & purificación , Persona de Mediana Edad , Orthomyxoviridae/genética , Orthomyxoviridae/inmunología , Cultivo Primario de Células , Mucosa Respiratoria/citología , Replicación Viral

RESUMEN

Previously unknown or unexpected pathogens may be responsible for that proportion of respiratory diseases in which a causative agent cannot be identified. The application of broad-spectrum, sequence independent virus discovery techniques may be useful to reduce this proportion and widen our knowledge about respiratory pathogens. Thanks to the availability of high-throughput sequencing (HTS) technology, it became today possible to detect viruses which are present at a very low load, but the clinical relevance of those viruses must be investigated. In this study we used VIDISCA-454, a restriction enzyme based virus discovery method that utilizes Roche 454 HTS system, on a nasal swab collected from a subject with respiratory complaints. A γ-papillomavirus was detected (complete genome: 7142 bp) and its role in disease was investigated. Respiratory samples collected both during the acute phase of the illness and 2 weeks after full recovery contained the virus. The patient presented antibodies directed against the virus but there was no difference between IgG levels in blood samples collected during the acute phase and 2 weeks after full recovery. We therefore concluded that the detected γ-papillomavirus is unlikely to be the causative agent of the respiratory complaints and its presence in the nose of the patient is not related to the disease. Although HTS based virus discovery techniques proved their great potential as a tool to clarify the etiology of some infectious diseases, the obtained information must be subjected to cautious interpretations. This study underlines the crucial importance of performing careful investigations on viruses identified when applying sensitive virus discovery techniques, since the mere identification of a virus and its presence in a clinical sample are not satisfactory proofs to establish a causative link with a disease.