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Applied and Environmental Microbiology, April 2008, p. 2544-2549, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02477-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Tracing of Norovirus Outbreak Strains in Mussels Collected near Sewage Effluents{triangledown}

Nancy P. Nenonen,1* Charles Hannoun,1 Peter Horal,1 Bodil Hernroth,2 and Tomas Bergström1

Department of Infectious Diseases/Clinical Virology, University of Gothenburg, Guldhedsgatan 10b, 413 46 Gothenburg, Sweden,1 The Royal Swedish Academy of Sciences, Sven Loven Centre for Marine Sciences, Kristineberg 566, SE-450 34 Fiskebäckskil, Sweden2

Received 2 November 2007/ Accepted 20 February 2008


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ABSTRACT
 
Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.


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INTRODUCTION
 
Noroviruses (NoV) are the most commonly identified cause of acute nonbacterial gastroenteritis in all age groups in the developed world (21). Symptoms are short lived, but virus levels in patient stools are high (12), and NoV is excreted directly into the sewage system (20). Although real-time studies (4) show improved water quality following sewage plant treatment, NoV breakthrough is common (24, 30, 32), particularly after heavy rainfall (23). The high prevalence of NoV genogroup I (GI) in wastewaters and treated effluents (4, 16) raises questions of environmental stability and resistance to wastewater treatment. Less is known about the circulation of NoV in other waters (22, 28, 30), although the importance of viral contamination from sewage effluents in shellfish growing areas is well recognized (2, 18).

The efficient filtering and concentrating capacities of filter-feeding bivalves (1, 11) support the use of these bivalves as sentinels for tracing NoV in polluted waters. Using bivalves to trace recent contamination with human sewage may provide information on NoV dispersal in effluents and coastal waters. To assess this potential, we examined wild mussels gathered close to the effluent plume from the Gothenburg sewage treatment plant, correlating the molecular findings with those of human strains from local outbreaks of acute gastroenteritis.


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Mussel samples.
 
Blue mussels (Mytilus edulis) were collected from Fotö Island in the northern archipelago, 13 km from the Rya wastewater treatment plant (RyaWWTP), the Gothenburg sewage station. Situated at the mouth of the Göta Álv River, the plant serves a population of 650,000. Fotö (population, 617) lies directly in the path of prevailing northerly currents carrying effluent plume from the RyaWWTP (7) and was therefore chosen for the NoV studies with wild mussels (Fig. 1). In November 2004, a batch of 40 mussels was collected and processed in the laboratory assigned for bivalve analysis, following the principles of Kwok and Higuchi (15). Each mussel was examined individually by emulsifying the whole digestive gland in 4 ml Hanks medium. Mussel gland suspensions aliquoted in 500-µl volumes were stored at –80°C. Suspensions (500 µl) were homogenized in 1,050 µl RNA III tissue lysis buffer (Roche Diagnostic GmbH, Germany) with a MagNA lyser instrument (Roche), using ceramic beads and oscillation (two 60-s cycles at 6,500 rpm). RNA was extracted from the tissue lysate (three 350-µl volumes), using a MagNA Pure LC instrument according to the RNA III protocol (elution volume, 50 µl), giving three extracts.


Figure 1
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  		FIG. 1. Diagram of the Gothenburg region showing the Göta Álv estuary, Fotö island, and the RyaWWWP, with the effluent plume represented by the arrow, and including Lake Delsjö and (in the neighboring Lerum region) Lake Aspen.


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Clinical samples.
 
Stool samples were obtained from symptomatic patients during a surveillance of community and institutional outbreaks of acute gastroenteritis in West Götaland. Samples emulsified as 10% suspensions in 4 ml Hanks medium were centrifuged at 700 x g for 7 min. Supernatants (130 µl) were added to MagNA Pure RNA III lysis buffer (220 µl), and RNA (100 µl) was extracted with the MagNA Pure LC instrument.


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NoV RNA detection and comparative analysis of RdRp.
 
Screening of RNA extracts was based on nested combined reverse transcription-PCR (RT-PCR) (25) of the NoV RNA-dependent RNA polymerase (RdRp) gene, using the primers (8, 17, 33, 35) and amplification programs outlined in Table 1. Serial dilutions (100, 10–1, and 10–2) of mussel RNA (10 µl) were amplified in NVp36/d110 RT-PCR master mixes (40 µl). Three RNA extracts were assayed. Water controls were included between samples (15). Amplified RT-PCR mixtures (5 µl and 8 µl) were nested into PCR master mixes (45 µl) containing JV12Y/JV13I primers. The specificities of gel-electrophoresed 328-bp products were confirmed by purification (25), cycle sequencing of both strands with a BigDye Terminator kit (version 1.1; Applied Biosystems, Foster City, CA), and sequence analysis on an Avant-3100 gene analyzer (Applied Biosystems, Foster City, CA). Three aliquots of each mussel suspension were homogenized and screened for NoV RdRp RNA.


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  		TABLE 1. Primers and RT-PCR conditions used in screening mussel digestive gland and patient stool samples for NoV RNA

In diagnostic testing of patient samples, RNA (10 µl) was added to three RT-PCR master mixes (40 µl). Each contained one forward primer (NI, NVp36, or NVp69) and the reverse primer NVp110 (Table 1). When amplicons were detected on gel electrophoresis of NI/NVp110 or NVp69/NVp110 RT-PCR mixtures, the NVp36/NVp110 RT-PCR mixture (5 µl) was selected for nesting into JV12Y/JV13I PCR master mixes (45 µl). Amplification gave standard-length 328-bp products for sequence confirmation and phylogenic analysis. Gel products, when detected in first-round NVp36/NVp110 RT-PCR mixtures, were sequenced with the respective primers.

NoV was detected in 23 of 40 mussels (57%), as confirmed by RdRp sequence analyses. Eighteen strains from mussels (n = 18) aligned with GI.1 reference sequences from GenBank, with one strain from another mussel aligning as GI.2. NoV GII strains were detected in four other mussels (two GII.2 and two GII.4 strains).

Mussel NoV RdRp sequences from bivalves collected in November were compared with patient outbreak sequences from the same year to trace the possible source of mussel contamination. NoV GI.1 strains from November mussels showed 100% sequence similarity (285 nucleotides [nt]) with GI.1 strains (n = 9) from Lake Delsjö bathers infected in August, earlier that year, during outbreaks associated with recreational bathing in inland lakes (27). More than 300 individuals were affected, and two distinct GI strains were identified in clinical samples examined in August, NoV GI.1 in Delsjö patients and GI.3 in Lake Aspen bathers (Fig. 1). None of the mussel strains aligned with GI.3 strains (n = 17) from Aspen bathers.


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Long-fragment amplification.
 
Comparative sequence analysis of RdRp-capsid-poly(A)-3' (3.1-kb) regions of NoV GI.1 strains from November mussels and Delsjö patients was used to trace the origins of mussel NoV strains. RNA from three mussel and five patient samples testing positive for RdRp was examined by seminested RT-PCR with primers as shown in Table 2 (6, 9, 19, 35). RT-PCR master mixes (25) were modified to include 0.4 mM betaine (Sigma-Aldrich), 5 units avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI), 1 unit AmpliTAQ DNA polymerase (Applied Biosystems), and NoV primers. Viral RNA was amplified from mussel extracts by RT-PCR with primers NVp36/N7610R, followed by two seminested PCRs using primer pairs NVp36/GV7 and GIFFN/N7610R. RNA extracts from patient samples were amplified by RT-PCR with primers NVp36/T25VN-3' and seminested PCR using NVp36/N7610R (Table 2). Purified gel products were cycle sequenced with primers giving contiguous sequences in both directions, as shown in Table 3 (6, 9, 12, 14, 17, 19, 33-35).


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  		TABLE 2. Primers and RT-PCR conditions used to amplify long-fragment NoV RdRp-capsid-poly(A)-3' regions from mussel and patient samples


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  		TABLE 3. Primers used in sequencing the 3.1-kb NoV region amplified from mussel and patient samplesa


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Gene studies and phylogenetic analyses.
 
Contiguous sequences of NoV GI.1 strains (n = 3) from November mussels showed 99.9% similarity, across 3,082 nt, with GI.1 strains (n = 5) from Delsjö patients infected the previous August. Mussel NoV strains differed from human strains at one position, where a synonymous change (C7444T) was noted (GenBank accession no. M87661). Identical sequences (3,082 nt) were obtained from three of the five Delsjö patients; sequences from the two other patients showed one synonymous change (nt position 7276). NoV sequences from mussels and patients were most similar to GI.1 reference sequences and showed unique GI.1 deletions in capsid amino acids 198 to 199, 289 to 299, 347 to 351, 375 to 377, and 442 to 445 compared with sequences from other GI subgroup strains. These gene study analyses indicate bioaccumulation of NoV strains of human origin in the mussels and provide evidence of the tracing of clinical strains in the environment.

Phylogenetic trees of 285- and 3,082-nt sequences were constructed based on neighbor-joining methods by using MacVector 7.2 software. The results confirmed the high similarity between NoV GI.1 strains from November mussels and those from Delsjö patients of August, as shown by clustering of strains in Fig. 2A and B. The GI.1 Delsjö strain showed 96% similarity with patient strain SRSV-KY-89/89/J, from a Japanese oyster outbreak in 1989 (GenBank accession no. L23828). Bootstrapping (1,000 replicates) gave reliable values of >75 on all nodes of constructed trees.


Figure 2
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  		FIG. 2. Neighbor-joining trees based on analysis of NoV RdRp (285 nt) (A) and NoV RdRp-capsid-poly(A)-3' (3,082 nt) (B). M identifies individual mussel strains. P represents patient strains linked to outbreaks. (n)* indicates the number of sequences analyzed and shown to be identical. Genotype subgroups are defined on branches beside GenBank accession numbers of reference strains. Bootstrap values of >75 are included. Genetic distance per nucleotide/site is shown by the bar.


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Significance of molecular findings.
 
We present molecular evidence of contaminant human NoV GI.1 RNA in wild mussels collected close to the effluent plume from a sewage treatment plant following major outbreaks of NoV GI.1 in the region. Sequence similarity between NoV GI.1 strains from mussels collected in November and from bathers sampled in August was high (>99.9%; 3,082 nt). With a 3-month time scale between the GI.1 Delsjö (August) outbreak and detection of NoV GI.1 in mussels sampled later that year (November), the sequence identity indicates the human origin of the mussel NoV strains. During that time period, heavy rainfall in Gothenburg was recorded (5), conditions associated with high concentrations of NoV in effluents from sewage plants (20, 32). Although it is unlikely, we cannot exclude the possibility that illicit waste dumping from recreational or commercial shipping was a cause of the gross mussel contamination (2).

These mussel findings suggest the environmental stability of NoV GI strains in water and bivalves (22, 26, 31). Recent European real-time studies show erratic peaking of GI strains in waters before and after sewage plant treatment, although NoV GI is rarely represented in medical reporting (4, 16, 24). In the present study, NoV GI.1 and GI.3 strains caused extensive outbreaks affecting children and adults, confirming the importance of GI infections in community outbreaks. The subsequent detection of NoV GI.1 (3.1 kb) in Fotö mussels collected more than 3 months after the Delsjö outbreak provides evidence of the stability of GI strains in bivalves and coastal waters.

This stability of the noncultivable NoV, combined with the unique filter-feeding habits of bivalves, may provide epidemiological information in "compacted time." Here, we focused on the potential of bivalve studies in tracing strains from past or recent-past NoV outbreaks circulating in a region, recording events that cannot be traced by direct examination of sewage effluents. Similar sentinel studies of hepatitis A virus in clams from Mozambique showed correlation with strains from patients of South and Northeast African origin (25). Bivalves, therefore, may prove a useful tool in tracing other viral agents.

In assessing molecular findings, the absence of Aspen NoV GI.3 strains from Fotö mussels at first seemed incongruous. This variant, causing outbreaks in neighboring Lerum, had circulated with GI.1 (Delsjö) during August (Fig. 1). However, wastewater from Lerum was processed not at the RyaWWTP but at a smaller plant with an outfall pipe close to the Aspen bathing area. As Lake Aspen lies approximately 34 km from Fotö (Fig. 1), disparity in regional population sizes (Gothenburg, 650,000; Lerum, 36,000) and factors such as distance, dilution, and rapid river flow (10) could explain the absence of Aspen strains in Fotö mussels. The negative NoV GI.3 (Aspen) findings further emphasize the significance of the GI.1 mussel findings from Fotö.

This study focused on tracing clinical strains in the environment, where environmental samples, mussels, were taken 3 months after the clinical samples were obtained from patients affected in extensive NoV outbreaks. To date, nucleotide information on NoV strains detected in shellfish has been limited to short sequences (≤400 nt), mainly from bivalves implicated in outbreaks (3, 13, 29). In contrast, here we have succeeded in sequencing 3.1-kb NoV regions from wild mussels collected close to the effluent plume from a sewage station by using long-fragment, classical nested RT-PCR methods in well-controlled systems (15). Resultant gene study analyses of mussel and outbreak strains provide evidence of bioaccumulation of NoV strains of human origin in the mussels.

We conclude that sentinel mussel studies can provide evidence of NoV circulation in coastal waters and contribute to our understanding of NoV epidemiology. Moreover, long-fragment analyses may provide an exploratory tool for studying virus-bivalve interactions.


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Nucleotide sequence accession numbers.
 
The sequences described in this study have GenBank accession numbers EU085483 to EU085490 and EU085497 to EU085529.


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ACKNOWLEDGMENTS
 
This work was supported by the Swedish International Development Cooperation Agency, Sida (grants SWE-2003-108 and 709 603), and the LUA-ALF Foundation (grant 7346), Sahlgren's University Hospital.

We thank Birgitta Bidefors, Mona Groot-Jensen, and members of the Department of Virus Detection, Virology, Sahlgren's University Hospital, Gothenburg, for skilled support and Jan Mattson from the RyaWWTP for advice on the hydrodynamics of the effluent plume.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases/Clinical Virology, University of Gothenburg, Guldhedsgatan 10b, 413 46 Gothenburg, Sweden. Phone: 46 736 601 740. Fax: 46 31827032. E-mail: nancy.nenonen{at}microbio.gu.se Back

{triangledown} Published ahead of print on 29 February 2008. Back


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Applied and Environmental Microbiology, April 2008, p. 2544-2549, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02477-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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