Molecular characterization of human adenoviruses in urban wastewaters using next generation and Sanger sequencing
Graphical abstract
Introduction
Human adenoviruses (HAdVs) are non-enveloped, icosahedral viruses characterized by a linear, double-stranded DNA genome. They belong to the family Adenoviridae of the genus Mastadenovirus. Using biological characteristics, 51 serotypes were identified and classified into seven species (A–G). Additional human adenovirus types were later identified based on genomic data, including several emerging and recombinant viruses (Ziros et al., 2015). The Human Adenovirus Working Group currently recognizes a total of 71 HAdV types having whole-genome sequence data.
Infections caused by HAdVs may be asymptomatic. Clinical manifestations are highly heterogeneous, ranging from upper and lower respiratory tract infections to gastroenteritis, pneumonia, urinary tract infection, conjunctivitis, hepatitis, myocarditis and encephalitis (Lion, 2014). HAdV species B1 (HAdV-B1), HAdV-C and -E tend to cause respiratory diseases; HAdV-B, -D and -E are mainly responsible for eye diseases; HAdV-B2 tends to infect kidneys and the urinary tract as well as the respiratory system; while HAdV-F mainly causes gastroenteritis (Russell, 2009). HAdVs have been associated with persistent infections in both immunocompetent and immunocompromised patients and can cause severe or life-threatening illness in children and the elderly (Lenaerts et al., 2008, Lion, 2014, Moyo et al., 2014).
Virtually all HAdV types, both enteric and non-enteric types, are excreted in high concentrations in the feces of infected patients (Akhter et al., 1995, La Rosa et al., 2015, La Rosa et al., 2006, La Rosa et al., 2011, Moyo et al., 2014), asymptomatic carriers included (Vetter et al., 2015).
The appearance of new variants and their severe morbidity, qualify HAdVs as emerging pathogens. This led to the establishment of specific surveillance programs, such as the Respiratory Adenovirus Surveillance in the USA (http://www.cdc.gov/surveillance/nrevss/adeno/index.html). In Italy, no nationwide surveillance system exists, and epidemiological data on HAdV infections is limited. In a previous study (La Rosa et al., 2011), we investigated types responsible for HAdV-related hospitalization in Italy, and confirmed species C, and particularly type 2 of this species, as one of the most frequently isolated HAdV from hospitalized patients. Overall, seven types belonging to three species (B, C and F) were identified in that study. Clinical surveillance misses mild, asymptomatic or subclinical infections, however. A more accurate picture of the types circulating in the community may be obtained through the study of viruses in sewage, and since different species and types have highly divergent pathogenic characteristics, information concerning their distributions would improve our understanding of water-related HAdV health risks.
In the present study we report the results of a one year survey of HAdVs in sewage samples collected from wastewater treatment plants (WTPs) located throughout Italy, by phylogenetic analysis of a segment of the hexon coding region of HAdVs. Previous investigations on adenoviruses in water environments mainly used direct Sanger sequencing of PCR products or sequencing of cloned PCR products into plasmids (Iaconelli et al., 2016, Amdiouni et al., 2012, Jiang et al., 2009, Kokkinos et al., 2011). The major disadvantages of this conventional sequencing approach is that only a limited number of representative sequences can be successfully obtained among the multiple viral strains potentially present in water samples. This may be due to the fact that, where more than one type is present in a sample, the broad-range assay successfully amplifies and sequences only the most prevalent type in terms of concentration, or those types that are preferentially amplified by the primers. Therefore, broad-range assays may grossly underestimate the prevalence of some, possibly less common, types, masked by the overwhelming presence of other strains. This, in turn, could also result in underestimates of overall HAdVs genetic diversity in environmental samples. Recently, next-generation sequencing (NGS) proved an effective tool for studying HAdV diversity in environmental samples (Bibby and Peccia, 2013, Ogorzaly et al., 2015). In the present study, we used both Sanger and next-generation sequencing methods, to provide a more complete picture of the distribution of HAdV types in urban wastewaters.
Section snippets
Samples
In total, we processed and analyzed 141 sewage samples from 22 wastewater treatment plants (WTPs) in 10 Italian regions, collected in 2013, in the framework of an existing network of WTPs, previously established for the surveillance of other enteric viruses (La Rosa et al., 2014, Muscillo et al., 2013). Fig. 1 shows a map with the location of the WTPs that participated in the present study.
Wastewater samples were collected, handled and analyzed as previously described (La Rosa et al., 2014).
Nested PCR and Sanger sequencing
A total of 141 sewage samples were tested for HAdVs by the nested PCR assays described above. Positive and negative controls yielded the expected results. Of the 141 tested samples, 85 (60%) tested positive by nested PCR. Molecular characterization of the sequences obtained by conventional Sanger sequencing of 59 amplicons showed the presence of a fragment of the hexon coding region of 4 HAdV species: A (type 12), B (type 3), C (type 5) or F (type 41). Adenovirus 41 was the most frequently
Discussion
Among the pathogenic agents transmitted via contaminated food or water, human enteric viruses are of particular concern due to their stability in water environments, high shedding concentrations and low infectious doses. Human enteric viruses infect the gastrointestinal tracts of infected individuals, replicate there and are released in large quantities in the hosts' stools (Okoh et al., 2010). Consequently, the discharge of inadequately treated sewage is a common source of enteric viral
Conclusion
As far as we know, this is the first study to test a large number of samples from numerous WTPs, and to demonstrate such a wide diversity of HAdV in urban sewage potentially able to impact other surface water environments and hence human health. Wastewater monitoring by NGS is expected to provide a more complete picture of the distribution of HAdV types in wastewaters, and, indirectly, of the circulation of HAdV types causing symptomatic and asymptomatic infections in the human population.
Acknowledgements
We thank Professor Herbert W. Virgin, Washington University (St. Louis, Missouri, United States) for providing the murine NoV strain used as sample process control.
We thank the assistance of the following members of the Italian Wastewater Network for wastewater sample collection: E. Lorenzi, M. De Ceglia (SMAT Spa, Castiglione T.se, Torino), L. Meucci, D. Giacosa, A. Poncino (Centro Ricerche SMAT, Torino); N. Mesiano (SMAT, Collegno); W. Bodini, C. Amadasi (Vettabbia Spa, Milano); F.
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2021, Water ResearchCitation Excerpt :Furthermore, we only performed classical Sanger sequencing on PCR amplicons, and this approach may underestimate the existence of some, possibly less common, sequences. Our next goal is therefore to combine the protocol with NGS on PCR amplicons for a more in-depth analysis of sequences, as successfully applied to enteric viruses (Suffredini et al., 2018; Iaconelli et al., 2017). The method can be used to discriminate the currently known SARS-CoV-2 variants, and may not be as useful with new variants possibly emerging in the future.
Next generation sequencing approaches to evaluate water and wastewater quality
2021, Water ResearchCitation Excerpt :Amplicon sequencing of 16S rRNA genes is often used to understand overarching shifts in microbial communities, such as disturbances, succession, and temporal trends. Similar approaches have also been used to identify waterborne eukaryotes (e.g., amoebae and protozoa) by targeting the 18S rRNA gene (Bradley et al., 2016), fungal communities by targeting the internal transcribed spacer (ITS) region (Bokulich and Mills, 2013), and specific virus families, for example by targeting the hexon gene specific to adenovirus (Iaconelli et al., 2017; Kuo et al., 2015). Commercial options are available that rely on this approach to target multiple gene targets simultaneously to profile multiple ARGs or pathogens, such as the AmpliSeq panels produced by Thermo Fisher Scientific.