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

Abstract

Background: Urban sewage virological analysis may produce important information about the strains that cause clinical and subclinical infections in the population, thus supporting epidemiological studies.

Methods: In the present study, a twenty one-month survey (November 2007 to July 2009) was conducted in order to evaluate the presence of human adenoviruses (hAdV), hepatitis A viruses (HAV), hepatitis E viruses (HEV), Noroviruses (NoV), and human Polyomaviruses (hPyV) in untreated sewage samples collected from the inlet of Patras' municipal biological wastewater treatment plant, located in southwestern Greece. Nucleic acid amplification techniques were applied for viral nucleic acid detection. Positive samples were confirmed by sequencing and comparative phylogenetic analysis was performed on the isolated viral strains.

Results: In total, viruses were detected in 87.5% (42/48) of sewage samples. AdVs, PyVs, HAV, and NoVs were detected in 45.8% (22/48), 68.8% (33/48), 8.3% (4/48), and 6.3% (3/48) of the samples collected from the plant's inlet, while HEV was not detected at all. Adenovirus types 8 (Ad8), 40 (Ad40) and 41 (Ad41) were recognized, while JC and BK polyomaviruses were recorded. Noroviruses were identified as GII.4. HAV was typed as genotype IA.

Conclusions: Our study demonstrates the advantages of environmental surveillance as a tool to elucidate the molecular epidemiology of community circulating viruses. We underline the need of environmental surveillance programs in countries such as Greece with inadequate and problematic epidemiological surveillance system and no environmental surveillance system currently in action.

Figure 1

Phylogenetic analysis of hAdV strains. An NJ phylogenetic tree was constructed to represent phylogenetic relationships among eighteen hAdV strains. Eleven strains (abbreviated as PAT73, PAT105, PAT274, PAT1413, PAT87, PAT86, PAT316, PAT486, PAT209, PAT689, PAT76) were isolated from Patras' sewage samples. Seven reference strains the sequences of which were retrieved from GenBank database were included to the analysis. Reference strains belong to hAdV genotype 8, 40, and 41. Simian AdV 22 strain [GenBank: AY530876] was used as an out-group. The bootstrap confidence levels were obtained for 1,000 replicates.

Figure 2

Phylogenetic tree of NoV strains. An NJ phylogenetic tree was constructed to represent phylogenetic relationships among three NoV strains of the present study (PAT1043, PAT105, PAT999) along with seven NoV reference strains. Numbers under branches are bootstrap percentage values, calculated from 1,000 bootstrap replicates. GenBank accession numbers and genotype of the reference sequences are included in the phylogram. Reference strains under the following accession numbers [GenBank: GU012345, AY772730, HM991391, HM802555, FJ537137, FJ905404, and EU794874], derived from Brazil, Germany, China, Hong Kong, USA, Tunisia and Belgium, respectively. The environmental reference NoV strain from Tunisy was isolated from treated sewage.

Figure 3

Phylogenetic tree constructed to represent phylogenetic relationships among eighteen HAV strains. Four HAV strains abbreviated as PAT73, PAT74, PAT76, PAT87 were isolated from Patras' sewage samples. Sixteen HAV references sequences were used for the phylogenetic tree constutcion. Nine reference strains corresponded to genotype IA [GenBank: AB020564, AB020568, AB020565, AB020569, EF207320, AF357222, AF512536, X75215], three to genotype IB [GenBank: M14707, M20273, AF314208], and one to genotype II [GenBank: M59810]. The bootstrap confidence levels obtained for 1,000 replicates are shown in the phylogram. Simian HAV strain [GenBank: D00924 genotype V] was used as an out-group.

Figure 4

Phylogenetic tree depicting the relationship between the environmental BK PyV strains of the present study compared with publicly available environmental BK sequences. Numbers under branches are bootstrap percentage values, calculated from 1,000 bootstrap replicates. GenBank accession numbers of the reference sequences are included in the phylogram. Strains abbreviated as PAT105, PAT125, PAT274, PAT486, PAT714 were isolated from raw sewage samples collected from Patras' biological treatment plant. Isolates env1, env2 and env3 were isolated from environmental water contaminated with sewage, while isolates SMM-1, SMM-2, and sew25 derived from raw sewage samples.

Figure 5

Phylogenetic tree depicting the relationship between the environmental JC PyV strains of the present study compared with publicly available environmental JC sequences. Nucleotide sequences of five Greek JC PyV strains isolated from raw sewage samples collected from the sewage treatment plant of the city of Patras (PAT, 71, PAT75, PAT635, PAT680, PAT700) were compared with fifteen reference environmental strains. GenBank accession numbers of the reference sequences are included in the phylogram. Reference strains abbreviated as PRETORIA1, USA2, E2, NANCY2, E1, G1, UMEA3, BCN15, DHR4a, RJ/2005-113, and LS1 were isolated from urban sewage derived from South Africa, United States, Egypt, Russia, Egypt, Greece, Czech Republic, United Kingdom, USA, Brazil, and USA respectively. Numbers under branches are bootstrap percentage values, calculated from 1,000 bootstrap replicates.