SORVEGLIANZA DELLE INFEZIONI RESPIRATORIE ACUTE (ARI): APPROCCI INNOVATIVI PER L'IDENTIFICAZIONE E LA CARATTERIZZAZIONE DEGLI AGENTI VIRALI COINVOLTI
Tesi di Dottorato
Data di Pubblicazione:
2014
Citazione:
SORVEGLIANZA DELLE INFEZIONI RESPIRATORIE ACUTE (ARI): APPROCCI INNOVATIVI PER L'IDENTIFICAZIONE E LA CARATTERIZZAZIONE DEGLI AGENTI VIRALI COINVOLTI / M. Martinelli ; tutor: E. Tanzi ; supervisori: L. van der Hoek, G. Zehender ; coordinatore: A. Zanetti. DIPARTIMENTO DI SCIENZE BIOMEDICHE PER LA SALUTE, 2014 Feb 26. 26. ciclo, Anno Accademico 2013. [10.13130/martinelli-marianna_phd2014-02-26].
Abstract:
Surveillance of acute respiratory infections (ARI): innovative approaches for the identification and characterization of viral agents
Introduction. Acute respiratory infections (ARI) are ubiquitous, air-borne transmitted and highly contagious infections, characterized by typical epidemic pattern. Though ARI causative agents may be bacterial or viral, viruses are by far the most common causes of ARI. In recent years, the global epidemiological scenario has been enlivened by the identification and emergence of many airborne pathogens (such as influenza viruses A/H5N1, A/H7N7, A/H7N9, coronaviruses SARS and MERS), which have been co-circulating along with the already known viral strains (i.e. seasonal influenza viruses, parainfluenza viruses, respiratory syncytial viruses, etc.), thus highlighting the public health concern about emerging infectious disease.
Objectives. The project aimed at applying new molecular biology techniques, "virus discovery" methodology and bioinformatics analyses to investigate the etiology of ARI.
The specific objectives were:
1) Identification of viral pathogens responsible of severe acute respiratory infections (SARI) and acute respiratory distress syndrome (ARDS) during the pandemic and post-pandemic (2009-2011). On purpose, the proportion of SARI/ARDS cases and deaths due to A(H1N1)pdm09 infection and the impact of other respiratory pathogens were evaluated during the pandemic and post-pandemic period in Lombardy. Additionally, unknown viruses were investigated in those cases for which diagnosis remained negative by using VIDISCA-454 methodology, a “virus discovery” technique. This analysis was performed at the Laboratory of experimental virology, Academic Medical Center (AMC), University of Amsterdam, where I completed an internship under the supervision of Prof. Lia van der Hoek.
2) Evaluating the genetic variability and molecular evolution of respiratory syncytial virus (RSV). In order to reconstruct the origin and phylodynamic history of RSV, the genetic diversity and evolutionary dynamics of RSV A and RSV B identified in respiratory specimens collected from children aged ≤ 3 years hospitalized in Lombardy for ARI over six epidemic seasons (2006 to 2012) were analyzed.
Materials and methods
1) From October 2009 to December 2011, 206 respiratory samples were collected from patients (61.2% males, median age: 44.3 years) hospitalized for SARI/ARDS. Nucleic acids were purified by NucliSENS® easyMAG® (bioMérieux, France), and analyzed by real-time RT-PCR assay to identify influenza virus. The clinical specimens that resulted negative to influenza virus detection were then screened by real-time RT-PCR/PCR for a panel of respiratory pathogens (Respiratory MWS r-gene™ Real-time PCR, bioMérieux, France) to detect: RSV A and B; human metapneumovirus (hMPV) A and B; human rhinovirus (hRV) and enterovirus (hEV); adenovirus (AdV); human bocavirus (hBoV) 1-4; human coronavirus (hCoV) 229E, NL63, OC43, HKU1; human parainfluenza virus (hPIV) 1-4; Chlamydophila pneumoniae; Mycoplasma pneumoniae. Cases resulted negative to all diagnostic assays were further investigated by VIDISCA-454 (virus discovery cDNA-AFLP) technique. This is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR combined with high-throughput sequencing 454 FLX/Titanium system of Roche.
2) RSV A (n=23) and RSV B (n=12) sequences obtained from oro-pharyngeal swabs of RSV-infected children aged ≤3 years hospitalized for ARI from 2006 to 2012 were considered for molecular characterization. Sequences were obtained by multiplex-PCR to amplify a fragment of RSV G gene and several bioinformatic programs were used for the phylogenetic and p
Tipologia IRIS:
Tesi di dottorato
Keywords:
acute respiratory distress syndrome (ARDS) ; influenza A(H1N1)pdm09 virus ; Respiratory viruses ; severe acute respiratory infection (SARI); VIDISCA-454 ; Respiratory Syncytial virus ; phylogenetic analysis
Elenco autori:
M. Martinelli
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