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Novel PHAge MEthods for improved virus inactivation

Periodic Reporting for period 1 - NoPHAME (Novel PHAge MEthods for improved virus inactivation)

Reporting period: 2019-01-01 to 2020-12-31

Infectious diseases can be spread through respiratory transmission (e.g. a sneeze spreading the common cold) or faecal-oral transmission (infected cook in restaurant uses a toilet without handwashing, contaminants food and the diners get infected). Diseases caused by faecal-orally transmitted viruses are major causes of illness globally. Infected individuals can produce and shed huge quantities of viruses into the sewage systems. Most of these viruses are very stable and can survive in sewage or on surfaces (e.g. door knobs) for days to months. Consumption of contaminated water or vegetables fertilized with contaminated water can lead to broad spread of the infections. Particularly, diarrhoea from contaminated drinking water causes more than 500,000 deaths annually, according to World Health Organization (WHO). Additionally, more than 2 billion people drink contaminated water daily and 2.4 billion lack access to safe toilets, resulting in further contamination of the water. While the burden of such diseases is higher in poorer countries, the safety of our water supply is challenged everywhere by rapid population growth, challenging the capacity of sewage treatment systems and resulting in increasing environmental pollution. Therefore, removal or inactivation of illness-causing viruses during wastewater treatment is a global issue. Diseases due to consuming contaminated water are preventable and the resulting deaths can be avoided. Because bacteria and protozoa are large and easy to detect, there are good methods for the removal of bacteria and protozoa. Methods for virus removal or inactivation is less advanced. The project develops the use of bacteriophages (viruses of bacteria) as powerful tools to monitor and improve virus inactivation and removal, resulting in cleaner and safer water. The novelty of this study is the combination of virology, metagenomics, deep sequencing, bioinformatics, and classical microbiology. In this fellowship, I have developed better systems to assess virus inactivation methods during wastewater treatment, through two objectives: (1) Identification of useful phages as markers or surrogates for pathogenic viruses; and (2) Development of phage replication assays to measure and quantify the phages during processing.
This research aimed to provide a more complete description of the bacteriophages (phages) present in sewage using primer-independent metagenomics sequencing approach. This primer-independent metagenomics sequencing approach combined with advanced computational method was employed to catalog and document all phages present in sewage samples from 2 sets of collection: the 81 global sewage samples collected from 62 countries around the world and then validated again in local Rotterdam sewage samples. Resulting data suggested a consistent set of phages that could be used as global/local phage markers, including phages in the Microviridae, Myoviridae, Podoviridae and Siphoviridae and Leviviridae. I have acquired classical phage handling methods including growth, purification, electron microscopy for visualisation. I have optimised effective phage sequencing and classification methods and I have developed a simple assay to monitor novel MS2 like phages. In addition to the novel phage knowledge I have generated, I have created a suite of phage tools and expertise and reagents that will support longer term projects for collaborators and myself beyond the period of my fellowship.
Peer-reviewed publications (10)
1. David Nieuwenhuijse*, My VT Phan*, Bas Oude Munnink*, et al. Setting a baseline for global urban virome surveillance in sewage. Scientific Reports. 2020 Aug 13;10(1):13748. doi: 10.1038/s41598-020-69869-0.
*These first authors contributed equally to the article.
2. Kirsty Kwok, Myrna de Rooij, Felisita F Sinartio, Lidwien AM Smit, Marion Koopmans, My VT Phan. Genome Sequence of a Minacovirus Strain from a Farmed Mink in the Netherlands. Microbial Resource Announcements. 2021 Feb 25;10(8):e01451-20. doi: 10.1128/MRA.01451-20.
3. Kirsty Kwok, Myrna de Rooij, Aniek Messink, Inge Wouters, Marion Koopmans, My VT Phan. Genome Sequences of Seven Megrivirus Strains from Chickens in the Netherlands. Microbial Resource Announcements. 2020 Nov 19;9(47):e01207-20. doi: 10.1128/MRA.01207-20
4. Rosa L. Allesøe and Camilla K. Lemvigh et al. Automated download and clean-up of family specific databases for kmer-based virus identification. Bioinformatics. 2020, Oct 8;btaa857. doi: 10.1093/bioinformatics/btaa857. Online ahead of print.
5. Gregorius J Sips et al. Norovirus outbreak in a natural playground: A One Health approach. Zoonoses and Public Health. 2020, 67(4):453-459. doi: 10.1111/zph.12689. Epub 2020 Feb 9.
6. Charles Masembe et al. Increased resolution of African Swine Fever Virus genome patterns based on profile HMMs of protein domains. Virus Evolution. 2020, Jun 19: veaa044.
7. Kirsty TT Kwok et al. Virus Metagenomics in Farm Animals: A Systematic Review. Viruses. 2020, 12(1):107. doi: 10.3390/v12010107.
8. Sofia Strubbia et al. Metavirome sequencing to evaluate norovirus diversity in sewage and related bioaccumulated oysters. Frontiers in Microbiology. 2019, 10: 2394. doi: 10.3389/fmicb.2019.02394. eCollection 2019.
9. Sofia Strubbia et al. Characterization of Norovirus and Other Human Enteric Viruses in Sewage and Stool Samples Through Next-Generation Sequencing. Food and Environmental Virology. 2019, 11(4):400-409. doi: 10.1007/s12560-019-09402-3. Epub 2019 Aug 24.
10. My VT Phan et al. Shedding of Yellow Fever Virus From an Imported Case in the Netherlands After Travel to Brazil. Open Forum Infectious Diseases. 2020, 7(2): ofaa020. doi: 10.1093/ofid/ofaa020.
Access to safe drinking water may be the largest problems facing the world. Contaminated water is a serious problem in developing countries and regions with dense population, poor sanitation and hygiene, and under-developed infrastructure. Sewage and wastewater management is complicated by incomplete knowledge about inactivation of illness-causing viruses during water treatment processes. The WHO has projected that by 2025, “half of the world population will be living in water-stressed areas”, and the related health consequences will be significant. Therefore, the results from this fellowship are expected to have potential global impact providing simple and easily implement virus inactivation/purification methods, will provide useful tools for improving wastewater management and water quality. This fellowship combines virology, bacteriology, bioinformatics and water engineering expertise. Because there is less research on RNA phages, this fellowship, with an emphasis on this class of phage, will provide novel insights that may be explored in future studies. The methods I established and quantitative assays I developed to measure the phage will provide important tools for research on infectious diseases in general and viral diseases in particular. Finally, the validation of water treatment technologies for RNA virus inactivation/purification can be deployed in developing countries where the need for clean water is greatest.
An illustration of plaque-forming bacteriophage isolated