Elsevier

Water Research

Volume 121, 15 September 2017, Pages 240-247
Water Research

Molecular characterization of human adenoviruses in urban wastewaters using next generation and Sanger sequencing

https://doi.org/10.1016/j.watres.2017.05.039Get rights and content

Highlights

  • Next generation (NGS) and Sanger sequencing to detect human adenovirus in wastewater.

  • NGS able to detect types unidentified by conventional Sanger sequencing.

  • A wide variety of human adenovirus species and types detected in raw sewage.

Abstract

Human adenoviruses (HAdVs) are of major public health importance and are associated with a variety of clinical manifestations, including gastroenteritis, respiratory, ocular and urinary tract infections.

To study the occurrence, prevalence and diversity of HAdV species and types circulating in Italy, we conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 22 wastewater treatment plants, covering 10 Italian regions, representative of northern, central, and southern Italy. A total of 141 raw sewage samples were collected from January to December 2013, and processed to detect and characterize by phylogenetic analysis a fragment of the hexon coding region of HAdVs.

Nested PCR results showed the presence of HAdVs in 85 out of 141 samples (60% of samples). Fifty-nine samples were characterized by conventional Sanger sequencing as belonging to four HAdV species and four types: A (type 12, 5 samples), B (type 3, 8 samples), C (type 5, 1 sample) and F (type 41, 45 samples). The remaining 26 samples could not be characterized because of uninterpretable (mixed) electropherograms suggesting the presence of multiple species and/or types.

Pools of characterized and uncharacterized PCR amplicons were further analyzed by next-generation sequencing (NGS). NGS results revealed a marked HAdV diversity with 16 additional types detected beyond the four types found by Sanger sequencing.

Overall, 19 types were identified, belonging to HAdV species A-F: types 12 and 31 (species A), type 3 (species B), types 1, 2, and 5 (species C), types 9, 17, 24, 26, 37, 38, 42, 44, 48, and 70 (species D), type 4 (species E), and types 40 and 41(species F). An untypeable HAdV was also detected, showing similar percentages of identity with more than one prototype (types 15, 30, 56, and 59).

Our findings documented the circulation of a wide variety of species and types in raw sewage, potentially able to affect other surface water environments and hence human health.

Next-generation sequencing proved to be an effective strategy for HAdV genotyping in wastewater samples. It was able to detect a wide range of “less prevalent” types unidentified by conventional Sanger sequencing, confirming that studies based on conventional technologies may grossly underestimate the existence of some, possibly less common, types.

Knowledge of the distribution of HAdV species and types would improve our understanding of waterborne HAdV-related health risks.

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.

References (52)

  • M. Avolio et al.

    When should adenoviral non-gonococcal urethritis be suspected? Two case reports

    New Microbiol.

    (2014)
  • K. Bibby et al.

    Identification of viral pathogen diversity in sewage sludge by metagenome analysis

    Environ. Sci. Technol.

    (2013)
  • S. Bofill-Mas et al.

    Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices

    Appl. Environ. Microbiol.

    (2006)
  • A. Carducci et al.

    Epidemiological surveillance of human enteric viruses by monitoring of different environmental matrices

    Water Sci. Technol.

    (2006)
  • A. Carraturo et al.

    Microbiological and epidemiological aspects of rotavirus and enteric adenovirus infections in hospitalized children in Italy

    New Microbiol.

    (2008)
  • S. Choi et al.

    Real-time PCR quantification of human adenoviruses in urban rivers indicates genome prevalence but low infectivity

    Appl. Environ. Microbiol.

    (2005)
  • E.R. Dubberke et al.

    Acute meningoencephalitis caused by adenovirus serotype 26

    J. Neurovirol.

    (2006)
  • C.A. Gaydos et al.

    Adenovirus vaccines in the U.S. military

    Mil. Med.

    (1995)
  • S. Hofmayer et al.

    Unique sequence features of the Human adenovirus 31 complete genomic sequence are conserved in clinical isolates

    BMC Genomics

    (2009)
  • M. Iaconelli et al.

    One-year surveillance of human enteric viruses in raw and treated wastewaters, downstream river waters, and drinking waters

    Food Environ. Virol.

    (2016)
  • H. Jalal et al.

    First reported outbreak of diarrhea due to adenovirus infection in a hematology unit for adults

    J. Clin. Microbiol.

    (2005)
  • S.C. Jiang et al.

    Evaluation of four cell lines for assay of infectious adenoviruses in water samples

    J. Water Health

    (2009)
  • M.S. Jones et al.

    Evaluation of type-specific real-time PCR assays using the LightCycler and J.B.A.I.D.S. for detection of adenoviruses in species HAdV-C

    PLoS One

    (2011)
  • A.E. Kajon et al.

    Molecular epidemiology of adenovirus type 4 infections in US military recruits in the postvaccination era (1997-2003)

    J. Infect. Dis.

    (2007)
  • D.S. Kelsey et al.

    Adenoviral meningoencephalitis in a patient with lead toxicity

    Arch. Neurol.

    (1979)
  • P.A. Kokkinos et al.

    Molecular detection of multiple viral targets in untreated urban sewage from Greece

    Virol. J.

    (2011)
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