Newly described human polyomaviruses Merkel Cell, KI and WU are present in urban sewage and may represent potential environmental contaminants

Recently, three new polyomaviruses (KI, WU and Merkel cell polyomavirus) have been reported to infect humans. It has also been suggested that lymphotropic polyomavirus, a virus of simian origin, infects humans. KI and WU polyomaviruses have been detected mainly in specimens from the respiratory tract while Merkel cell polyomavirus has been described in a very high percentage of Merkel cell carcinomas. The distribution, excretion level and transmission routes of these viruses remain unknown

Characterisation of the sewage virome: comparison of NGS tools and occurrence of significant pathogens

NGS techniques are excellent tools to monitor and identify viral pathogens circulating among the population with some limitations that need to be overcome, especially in complex matrices. Sewage contains a high amount of other microorganisms that could interfere when trying to sequence viruses for which random PCR amplifications are needed before NGS. The selection of appropriate NGS tools is important for reliable identification of viral diversity among the population. We have compared different NGS methodologies (Untargeted Viral Metagenomics, Target Enrichment Sequencing and Amplicon Deep Sequencing) for the detection and characterisation of viruses in urban sewage, focusing on three important human pathogens: papillomaviruses, adenoviruses and enteroviruses. A full picture of excreted viruses was obtained by applying Untargeted Viral Metagenomics, which detected members of four different vertebrate viral families in addition to bacteriophages, plant viruses and viruses infecting other hosts. Target Enrichment Sequencing, using specific vertebrate viral probes, allowed the detection of up to eight families containing human viruses, with high variety of types within the families and with a high genome coverage. By applying Amplicon Deep Sequencing, the diversity of enteroviruses, adenoviruses and papillomaviruses observed was higher than when applying the other two strategies and this technique allowed the subtyping of an enterovirus A71 C1 strain related to a brainstem encephalitis outbreak occurring at the same time in the sampling area. From the data obtained, we concluded that the different strategies studied provided different levels of analysis: TES is the best strategy to obtain a broad picture of human viruses present in complex samples such as sewage. Other NGS strategies are useful for studying the virome of complex samples when also targeting viruses infecting plants, bacteria, invertebrates or fungi (Untargeted Viral Metagenomics) or when observing the variety within a sole viral family is the objective of the study (Amplicon Deep Sequencing)

Viral metagenomics reveals persistent as well as dietary acquired viruses in Antarctic fur seals.

Viruses linked to animals inhabiting Antarctic latitudes remain poorly studied. Remote environments hosting large pinniped populations may be prone to exposure of immunologically naïve animals to new infectious agents due to increasing human presence or introduction of new animal species. Antarctic fur seals (Arctocephalus gazella) inhabiting the Western Antarctic Peninsula and the South Shetland Islands are challenged because of climate change and increased anthropogenic activity. In the present study, the fecal and serum virome of A. gazella was characterized by applying target enrichment next generation sequencing. The resulting viromes were dominated by CRESS-DNA sequences. Viruses known to infect vertebrate and invertebrate hosts were also observed in fecal samples. Fur seal picornavirus was present in all the fecal pools studied suggesting it is a prevalent virus in these species. Six different viruses presenting similarities with previously described A. gazella viruses or other otariids and mammal viruses were identified as potential new A. gazella viruses. Also, diet-derived viruses such as crustacean viruses were present in fecal content. Penguin viruses, but not fish viruses, were also detected. Obtained results contribute to a better understanding of the viral community present in these species, which is relevant for its conservation

The unexplored virome of two Atlantic coast fish: contribution of next-generation sequencing to fish virology

Much of the knowledge on viruses is focused on those that can be propagated using cell-cultures or that can cause disease in humans or in economically important animals and plants. However, this only reflects a small portion of the virosphere. Therefore, in this study, we explore by targeted next-generation sequencing, how the virome varies between Atlantic horse mackerels and gilthead seabreams from fisheries and aquaculture from the center and south regions of Portugal. Viral genomes potentially pathogenic to fish and crustaceans, as well as to humans, were identified namely Astroviridae, Nodaviridae, Hepadnaviridae, Birnaviridae, Caliciviridae, and Picornaviridae families. Also bacteriophages sequences were identified corresponding to the majority of sequences detected, with Myoviridae, Podoviridae, and Siphoviridae, the most widespread families in both fish species. However, these findings can also be due to the presence of bacteria in fish tissues, or even to contamination. Overall, seabreams harbored viruses from a smaller number of families in comparison with mackerels. Therefore, the obtained data show that fish sold for consumption can harbor a high diversity of viruses, many of which are unknown, reflecting the overall uncharacterized virome of fish. While cross-species transmission of bonafide fish viruses to humans is unlikely, the finding of human pathogenic viruses in fish suggest that fish virome can be a potential threat regarding food safety

Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas

Molecular techniques and virus concentration methods have shown that previously unknown viruses are shed by humans and animals, and may be transmitted by sewage-contaminated water. In the present study, river water from urban areas in Barcelona, Spain and Rio de Janeiro, Brazil, were analyzed to evaluate the dissemination of human viruses, while simultaneously optimizing and validating a low-cost concentration method for virus quantification in fresh water. The following three viral groups were analyzed. (i) Recently described viruses: klassevirus (KV), asfarvirus-like virus (ASFLV), and the polyomaviruses Merkel cell, KI and WU (MCPyV/KIPyV/WUPyV). (ii) Gastroenteritis agents: noroviruses (NoV) and rotaviruses (RV). (iii) Human fecal viral indicators in water: human adenoviruses (HAdV) and JC polyomaviruses (JCPyV). Virus detection was based on nested and quantitative PCR assays. Nested PCR assays were developed for KV and ASFLV. The method applied for virus concentration in water samples was a one-step procedure based on a skimmed milk flocculation procedure described previously for seawater. Using spiked river water samples, inter- and intra-laboratory assays showed a viral recovery rate of about 50% for HAdV, JCPyV, NoV and RV with a coefficient of variation ≤ 50%. HAdV and JCPyV were detected in 100% of the river samples from Barcelona and Rio de Janeiro. Moreover, NoV GGII was detected in 100% and MCPyV in 50% of the samples from Barcelona, whereas none of the other viruses analyzed were detected. NoV GGII was detected in 33%, KV in 33%, ASFLV in 17% and MCPyV in 50% of the samples from Rio de Janeiro, whereas KIPyV and WUPyV were not detected. RV were only tested for in Rio de Janeiro and resulted positive in 67% of the samples. The procedure applied here to river water represents a useful, straightforward and cost-effective method that could be applied in routine water quality testing.  The results of the assays expand our understanding of the global distribution of the viral pathogens studied here and their persistence in the environment. Fil: Calgua, B.. Universidad de Barcelona; España;Fil: Fumian, T.. Ministerio de Salud de Brasil. Fundacion Oswaldo Cruz; Brasil;Fil: Rusinol, M.. Universidad de Barcelona; España;Fil: Rodríguez Manzano, J.. Universidad de Barcelona; España;Fil: Mbayed, Viviana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Bofill Mas, S.. Universidad de Barcelona; España;Fil: Miagostovich, M.. Ministerio de Salud de Brasil. Fundacion Oswaldo Cruz; Brasil;Fil: Girones, R.. Universidad de Barcelona; España

Unveiling Viruses Associated with Gastroenteritis Using a Metagenomics Approach

Acute infectious gastroenteritis is an important illness worldwide, especially on children, with viruses accounting for approximately 70% of the acute cases. A high number of these cases have an unknown etiological agent and the rise of next generation sequencing technologies has opened new opportunities for viral pathogen detection and discovery. Viral metagenomics in routine clinical settings has the potential to identify unexpected or novel variants of viral pathogens that cause gastroenteritis. In this study, 124 samples from acute gastroenteritis patients from 2012-2014 previously tested negative for common gastroenteritis pathogens were pooled by age and analyzed by next generation sequencing (NGS) to elucidate unidentified viral infections. The most abundant sequences detected potentially associated to acute gastroenteritis were from Astroviridae and Caliciviridae families, with the detection of norovirus GIV and sapoviruses. Lower number of contigs associated to rotaviruses were detected. As expected, other viruses that may be associated to gastroenteritis but also produce persistent infections in the gut were identified including several Picornaviridae members (EV, parechoviruses, cardioviruses) and adenoviruses. According to the sequencing data, astroviruses, sapoviruses and NoV GIV should be added to the list of viral pathogens screened in routine clinical analysis

NGS techniques reveal a high diversity of RNA viral pathogens and papillomaviruses in fresh produce and irrigation water

Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samples

Development of improved low-cost ceramic water filters for viral removal in the Haitian context

Household-based water treatment (HWT) is increasingly being promoted to improve water quality and, therefore, health status in low-income countries. Ceramic water filters (CWFs) are used in many regions as sustainable HWT and have been proven to meet World Health Organization (WHO) microbiological performance targets for bacterial removal (24 log); however, the described viral removal efficiencies are insufficient to significantly reduce the associated risk of viral infection. With the objective of improving the viral removal efficiencies of ceramic water filters, new prototypes with different oxide compositions and firing atmospheres have been developed and evaluated. For removal efficiencies human adenoviruses, MS2 bacteriophage and Escherichia coli were quantified in all prototypes. A new model of CWF that was fired in a reductive atmosphere presented virus and bacteria removal efficiencies greater than 3.0 log and 2.5 log, respectively, which would fulfill the viral targets that are recommended by the WHO. Ceramic characterization of the selected filters, which were fired in a reductive atmosphere, showed that a larger specific surface area than those of control filters and higher fraction of a positive Z-potential fraction are the most likely explanations for this increase in virus removal

Metagenomics for the study of viruses in urban sewage as a tool for public health surveillance

The application of next-generation sequencing (NGS) techniques for the identification of viruses present in urban sewage has not been fully explored. This is partially due to a lack of reliable and sensitive protocols for studying viral diversity and to the highly complex analysis required for NGS data processing. One important step towards this goal is finding methods that can efficiently concentrate viruses from sewage samples. Here the application of a virus concentration method based on skimmed milk organic flocculation (SMF) using 10 L of sewage collected in different seasons enabled the detection of many viruses. However, some viruses, such as human adenoviruses, could not always be detected using metagenomics, even when quantitative PCR (qPCR) assessments were positive. A targeted metagenomic assay for adenoviruses was conducted and 59.41% of the obtained reads were assigned to murine adenoviruses. However, up to 20 different human adenoviruses (HAdV) were detected by this targeted assay being the most abundant HAdV-41 (29.24%) and HAdV-51 (1.63%). To improve metagenomics' sensitivity, two different protocols for virus concentration were comparatively analysed: an ultracentrifugation protocol and a lower-volume SMF protocol. The sewage virome contained 41 viral families, including pathogenic viral species from families Caliciviridae, Adenoviridae, Astroviridae, Picornaviridae, Polyomaviridae, Papillomaviridae and Hepeviridae. The contribution of urine to sewage metavirome seems to be restricted to a few specific DNA viral families, including the polyomavirus and papillomavirus species. In experimental infections with sewage in a rhesus macaque model, infective human hepatitis E and JC polyomavirus were identified. Urban raw sewage consists of the excreta of thousands of inhabitants; therefore, it is a representative sample for epidemiological surveillance purposes. The knowledge of the metavirome is of significance to public health, highlighting the presence of viral strains that are circulating within a population while acting as a complex matrix for viral discovery. (c) 2017 Elsevier B.V. All rights reserved