Seqüenciació massiva aplicada a l'epidemiologia d'aigües residuals i a la caracterització de viromes

Abstract

L’objectiu principal de la present tesi doctoral ha estat l’optimització i aplicació de procediments basats en la seqüenciació massiva per a la descripció del viroma associat a l’aigua residual i a altres mostres d’interès epidemiològic.

Els objectius específics han estat:

1. Optimitzar i aplicar diferents tècniques de seqüenciació massiva per a l’anàlisi de mostres ambientals i biològiques.

2. Caracteritzar el viroma de l’aigua residual amb focus en els patògens humans.

3. Avaluar l’ús del la seqüenciació massiva, amb i sense enriquiment de dianes, i de la seqüenciació d’amplicons per a l’estudi i monitoreig de brots infecciosos d’origen viral.

4. Aplicar la seqüenciació massiva a mostres de pacients amb gastroenteritis d’etiologia desconeguda per conèixer la presència d’espècies virals no detectades pels mètodes de diagnòstic clàssics.

5. Estudiar el viroma de diverses espècies de peixos Atlàntics amb potencials implicacions comercials i de seguretat alimentària.

Next generation sequencing (NGS) techniques have emerged in the last decade as keystone for the thorough study of microorganisms in a wide variety of samples and settings, replacing traditional molecular methods. In the field of virology, the constant evolution of sequencing platforms and applications enabled the improvement of virome studies. The main limitation when analysing the virome from any type of sample is the low proportion of viral sequences identified compared with the total number of sequences, especially critical for human viruses.

In this work we aimed to evaluate the use of different sequencing approaches, target enrichment (TES) and amplicon deep sequencing (ADS), for the characterization of the virome and specific viral pathogens in sewage and as tools for efficient wastewater-based epidemiology in outbreak scenario. The application of TES has proved to be a very successful strategy for the study of vertebrate viruses in sewage samples providing a higher number of detected families, a higher number of members within these families, more reads and larger genome coverage than conventional untargeted viral metagenomics. Additionally, allowed the obtention of SARS-CoV-2 sequences as part of sewage virome in a COVID-19 pandemic context, retrieving also other relevant human and animal coronavirus sequences, shedding light on the co-circulation of different strains in a determined population. In contrast, ADS proved to a very sensitive technique for the description of the diversity within a viral family, enabling the subtyping of sequences belonging to Enterovirus A71 C1 in sewage, while an encephalitis outbreak caused by this strain was happening during sampling period.

NGS, with and without TES panels, was also evaluated for the study of viral etiological agents of acute gastroenteritis in a collection of samples tested negative for the commonly associated pathogens. Its application resulted in the detection of emergent viral variants, like Norovirus GIV, and viruses not traditionally tested, like sapoviruses and astroviruses. These results highlighted the need of the incorporation of these viruses in clinical testing and the potential use of viral metagenomics as a diagnostic tool.

Lastly, to evaluate the use of enrichment panel in animal virology, TES was applied for the virome study of two economically important fish species from the Portuguese Atlantic coast. Pathogens causing viral nervous necrosis and infectious pancreatic necrosis in fishes were detected, demonstrating the utility of NGS techniques for the study of infections that may cause an economic impact in fish industry. Also, the identification of human noroviruses sequences in one of the fish samples suggested that fish virome studies can be used for evaluating potential threats regarding food safety.