Rotavirus (RV) and norovirus (NoV) are major causes of pediatric diarrhea and are altogether associated with approximately 800,000 deaths in young children every year. In Nicaragua, national RV vaccination program using the pentavalent RV5 vaccine from Merck was implemented in October 2006. To determine whether RV vaccination decreased the overall number of RV infections, we investigated the occurrence of RV and NoV in wastewater in the city of León from July 2007 to July 2008 and compared these data with pre-vaccination data. The major finding was the low prevalence of RV compared to NoV in all sampling points (11% vs 44%, p<0.05), and that RV concentration was lower as compared to NoV. RV was observed mainly during the rainy season (July–September), and the majority of all RV detected (6/9) belonged to subgroup (SG) I. The partial VP7-gene obtained from one RV positive sample was similar (99% nt identity) to a G6 VP7-gene of bovine origin and similar to the corresponding gene of the vaccine strain (98%). Furthermore RV G-types 2 and 4 were found in the incoming wastewater. NoV strains were detected throughout the year, of which a majority (20/21) were of genotype GII.4. We conclude that the introduction of RV vaccination reduced the transmission of RV in the community in Nicaragua. However, the burden of diarrhea in the country remains high, and the high prevalence of NoVs in hospital and municipal wastewater is noteworthy. This study highlights the need for further assessment of NoV following RV vaccine introduction.
Citation: Bucardo F, Lindgren P-E, Svensson L, Nordgren J (2011) Low Prevalence of Rotavirus and High Prevalence of Norovirus in Hospital and Community Wastewater after Introduction of Rotavirus Vaccine in Nicaragua. PLoS ONE 6(10): e25962. https://doi.org/10.1371/journal.pone.0025962
Editor: Karol Sestak, Tulane University, United States of America
Received: June 29, 2011; Accepted: September 14, 2011; Published: October 7, 2011
Copyright: © 2011 Bucardo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants from the Swedish International Development Cooperation Agency (http://www.sida.se/English/) (grant 0.75007109) and by the Swedish Research Council (http://www.vr.se/) (grant 10392). Dr. Bucardo received a travel grant from NETROPICA (http://www.netropica.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Rotavirus (RV) causes approximately 111 million episodes of diarrhea and is associated with 527,000 deaths in young children every year, predominantly from developing countries. Norovirus (NoV) causes 900,000 clinical visits in industrialized countries and up to 200,000 deaths in children from developing countries , , . Rotavirus is classified in serogroups (A–G), and based on molecular analysis of VP6 encoding gene, group A RV are divided in subgroups (SGI and SGII) , . After safety and efficacy data evaluation of the two licensed RV vaccines (Rotarix and RotaTeq) the World Health Organization (WHO) strongly recommended the inclusion of RV vaccines in the Expanded Program of Immunization (EPI) of vulnerable countries . From October 2006, the RotaTeq vaccine from Merck (RV5) is administered to all Nicaraguan children that are age-eligible.
RV and NoV typically infect the cell lining of the gastrointestinal tract and are discharged in very large numbers in the feces of symptomatic and asymptomatic infected persons (>1×108 virus particles per grams of feces) , , . Several reports indicate that both viruses are frequently found in water contaminated with human feces and detection rates follow a temporal pattern , , , , . Viral concentration of RV and NoV can range between 1×104 and 2×107 genome equivalents per liter (g.e l−1) of raw wastewater , , .
By measuring enteric viral levels and determining the corresponding genotypes in wastewater samples, it is possible to indirectly monitor the presence of these enteric agents in the community. Previous studies have found a correlation between enteric viruses in wastewater and viruses causing disease in the community , , . Furthermore, these types of investigations enable a more complete view of virus diversity at community level by also detecting viral strains that give rise to mild or asymptomatic infections, which are generally not observed at the clinical level , .
In this study, our objective was to determine whether the introduction of the RV5 vaccine in Nicaragua had reduced the circulation of RV in the community, and if shedding of RV vaccine strains had occurred into wastewater. Furthermore, we wanted to determine the temporal circulation of NoV and RV during one-year in Nicaragua after RV vaccination. This was carried out by performing a follow-up study of these viruses in sewage from the local hospital and the main wastewater treatment plant (WWTP) in the city of León after introduction of the pentavalent RV vaccine (RV5) in the EPI in Nicaragua.
In this study RV and NoV prevalence in wastewater was investigated in a country of Central America which has implemented RV vaccination in its national immunization schedule. This is also the first study to characterize enteric viruses in wastewater in Central America.
Materials and Methods
This study was carried out in the city of León, located close to the pacific cost of Nicaragua with an estimated population of 200,000 inhabitants. The climate is tropical; the rain season starts in June and lasts until November, when the dry season starts. Nicaragua was the first GAVI-eligible country to introduce rotavirus vaccination in October 2006. The Merck RV5 vaccine is provided to all Nicaraguan children that are age-eligible .
Description of the wastewater treatment plant (WWTP)
The wastewater treatment plant was designed for clarification and sedimentation of wastewater. In the first step solids are removed by filtration. In the second step, large basins hold wastewater for a period of time, allowing soil sediments or other materials suspended in water to settle at the bottom. The last step is to discard the clarified water into the local river (Rio Chiquito). This WWTP receives wastewater from approximately 70% of the households of León, neighborhoods along the river including the historical center and the “Hospital Escuela Oscar Danilo Rosales” (HEODRA). During the dry and rainy seasons approximately 235 l/s and 336 l/s pass through the WWTP, respectively (personal communication, Lic. Maria José Diaz). The clarified water discharged by the WWTP eventually reaches the Pacific Ocean through an estuary located at the pacific coast (Las Peñitas) of Nicaragua.
From July 2007 to July 2008 a total of 84 wastewater samples were collected from the inflow and outflow of the WWTP as well as from the wastewater drainer of the hospital (HEODRA). All necessary permits were obtained for the described field studies by the Director of HEODRA and the responsible for water affairs (ENACAL) in León. Weekly sampling was carried out every Thursday morning, from July to December 2007, to cover the rainy season, which coincides with the yearly epidemic peak of diarrhea after the RV vaccine introduction . Monthly sampling (n = 1) was then carried out the second Thursday of each month from February until July 2008. No samples were collected in January 2008 due to logistic constrains. A grab sample was collected from the sewage drainer of the hospital, where wastewater can be collected before reaching the public system, between 7 and 8 AM (high flow). Then, between 9 and 10 AM, the same day 2 grab samples were collected from the inflow (before the mechanical filter) and outflow of the WWTP, respectively. At each site, 50 ml of sewage water was collected in sterile BD Falcon™ conical tubes (Becton, Dickinson and Company), and transported at 4°C to the microbiology laboratory of UNAN-León. Samples were then frozen at −20°C and transported to Linköping University, Sweden, for further analysis. Prior to sample collection, temperature and pH were registered. To avoid carryover contamination, tubes containing the samples were not opened until ultracentrifugation.
Sample clarification and virus concentration
Before processing, samples were thawed overnight at 4°C, and clarified by centrifugation at 3,500× g for 5 min at 4°C. A total of 38 ml of supernatant were transferred to a thin wall, Ultra-Clear™ ultracentrifugation tube (Beckman, 344058). Ultracentrifugation was carried out in the Beckman Coulter - Optima™ L-100XP preparative ultracentrifuge (Beckman Coulter, Inc, CA, USA) using a SW 32 Ti Rotor at 164,000 g for 2 hours. A total of 37.5 ml of the supernatant was discarded by pipetting and the pellet was re-suspended in 0.5 ml of supernatant and frozen at −20°C until viral RNA purification. Distilled water was used as negative control in every round of centrifugation.
Viral RNA was extracted from 140 µl of the diluted pellet, following the manufacturer's instructions using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). A total of 60 µl of viral RNA was collected and stored at −70°C until reverse transcription (RT).
RT was essentially carried out as described previously , . Briefly, 28 µl of RNA was mixed with 50 pmol of random hexadeoxynucleotides [pd(N)6] (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), denatured at 97°C for 5 min, and quickly chilled on ice for 2 min, followed by addition of one RT-PCR bead (GE Healthcare Bio-Sciences AB) and RNase-free water to a final volume of 50 µl. The RT reaction was carried out for 30 min at 42°C to synthesize the cDNA used for screening and quantification of RV and NoV.
Detection and quantification of RV and NoV
RV was detected, subgrouped and quantified using a real-time PCR assay . The assay can subgroup RV strains based on subgroup specific fluorogenic primers targeting the VP6-gene, and semi-quantify using external plasmid standards.
NoV detection and quantification were performed with a real-time PCR targeting the ORF1-ORF2 junction, as described by Nordgren and co-workers . This real-time PCR assay can semi-quantify and distinguish between NoVs GI and GII .
Determination of NoV and RV concentrations and reduction
By using a standard curve in each real-time PCR assay as described elsewhere , , the number of viral genomes per real-time PCR reaction was determined, thus allowing us to estimate the number of both NoV and RV particles per liter of wastewater. The lowest detection level of NoV and RV in the real-time PCR assay is theoretically 1 gene per PCR reaction which corresponds to 2.5×103 genome equivalents (g.e.) per liter (l−1) of H2O using the methodology described in this paper. Furthermore, quantification of concentrations lower than 2.5×104 g.e. l−1 H2O (10 genes per PCR reaction) is less accurate due to less linearity of the real-time PCR standard curve close to the detection limit. The reduction of virus in the wastewater treatment plant was determined as the log10 transformation of the quotient of amount of virus before treatment, and the amount of virus after treatment. Due to the limited detection of RV in wastewater, the reduction was only determined for NoV. When NoV was below the detection limit in the outgoing water, a hypothetical value of 1.25×103 g.e. l−1 H2O (which corresponds to approximately half of the detection limit) was given for measurement of average reduction levels , .
RV VP7 genotyping and sequencing
A PCR mix was prepared by mixing 2.5 µl of cDNA, 4 pmol of each consensus primer (VP7-F and VP7-R) , 20.5 µl of RNase-free water and a PuReTaq Ready-To-Go PCR bead (GE Healthcare Bio-Sciences AB). PCR was performed at 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, with a final extension of 72°C for 7 min. The VP7 PCR-product was stored at −20°C until VP7 genotyping. A PCR mix was prepared by mixing 1 µl of VP7 PCR product, 4 pmol of each G genotype-specific and consensus primers (G1, G2, G3, G4, G8, G9 and VP7-R) , 23 µl of RNase-free water and a PuReTaq Ready-To-Go PCR bead (GE Healthcare Bio-Sciences AB). PCR was performed at 94°C for 4 min, followed by 30 cycles of 94°C for 1 min, 42°C for 2 min, and 72°C for 1 min, with a final extension of 72°C for 7 min. The PCR products were analyzed using 2% agarose gel and ethidium bromide staining, VP7 genotypes were assigned as described elsewhere .
The VP7 gene from one sample, containing sufficient RV concentration to enable an amplification of the VP7-gene, was sequenced using BigDye chemistry at Macrogen Inc. (Seoul, South Korea), with the primers VP7-F and VP7-R  as sequencing primers.
PCR amplification and cloning of the NoV N-terminal and shell (NS) region
PCR amplification of the NS region was performed using the forward primer  and reverse primer  as described elsewhere . The NoV NS capsid fragment was cloned into pJET1.2 vector and transformed into E.coli cells using CloneJet™ PCR Cloning and TransformAid™ Bacterial Transformation kits (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. Ten separate colonies from each transformation reaction were screened by PCR for the correct gene insert. After overnight incubation of positive colonies, plasmid DNA was extracted and purified using GeneJET™ Plasmid Miniprep Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. Nucleotide sequencing was performed by Macrogen Inc. (Seoul, South Korea), using the BigDye chemistry with pJET1.2 forward and reverse sequencing primers.
Genotyping of NoV by sequence analysis of the NS region
Sequence alignment of the Nicaraguan strains and reference NoV genotypes representing known genotypes as well as GII.4 variants, was performed by using the ClustalW algorithm, version 2, at the European Bioinformatics Institute server (EMBL-EBI). Phylogenetic analysis was performed using the MEGA 5.0 software package, and the tree was constructed using the neighbor-joining and Kimura two parameter methods. Significance of the relationship was obtained by bootstrap re-sampling analysis (1,000 replications).
Nucleotide accession numbers
The nt sequences as used for phylogenetic analysis (nt position 5085 to 5366; Ref acc.no X86557) of the norovirus NS region have been deposited in GenBank under accession numbers JN102111–JN102131.
Low prevalence of RV and high prevalence of NoV in hospital and community wastewater
All wastewater samples from July 2007 to July 2008 were tested for RV and NoV using sensitive real time-PCR methods ,  and the results demonstrated RV and NoV in 9 (11%) and 37 (44%) of the 84 sewage samples, respectively. Of the 9 RV-positive samples, 7 were also positive for NoV. The prevalence of RV in samples collected at the hospital wastewater, WWTP-inflow and WWTP-outflow were 7% (2/28), 21% (6/28) and 4% (1/28), respectively. In contrast, a higher NoV prevalence was observed at all sampling points, with 46% (13/28) in the hospital water, 54% (15/28) at the WWTP-inflow and 32% (9/28) at the WWTP-outflow (p<0.05; for all sampling points) (Table 1).
Seasonality of RV and NoV in the wastewater samples
This epidemiological parameter was investigated in the samples collected at the inflow of the WWTP, as they best represent the viruses circulating in the community. RV-positive samples were observed in July (2/4), August (2/4) and September (1/4) of 2007, and disappeared in the following months of surveillance, except in February (1/1) of 2008 (Table 1). An interesting observation was that RV seemed to circulate in the community a few weeks in July and August before its appearance in the hospital sewage water in September 2007 (Table 1). While RV circulated in very short time frames, NoV circulated year around, and only in the months of October of 2007 and April–May 2008 was it absent in wastewater (Table 1). NoV seemed to exhibit two periods of high concentration in incoming wastewater, during the rainy season (July to September), and during the winter season (November to March) while RV was mainly detected during the rainy season (July to September 2007) (Table 1).
Higher concentrations of NoV compared to RV in hospital and community wastewater samples
To investigate if RV and NoV quantities in the sewage were related to frequency, time and place of collection, we measured viral concentrations for NoV and RV in all sampling and time points. The frequency of RVs was low as compared to NoVs, which was also reflected in the quantification data (Fig. 1). The concentration of NoV ranged between 1.3×104 to 5.7×106 g.e. l−1 H2O. The highest concentration of NoV was found in hospital sewage in September 2007 (2.6×106 g.e. l−1 H2O) and in the inflow to the WWTP in December 2007 (5.7×106 g.e. l−1 H2O). The concentration of RV ranged between 1.3×104 and 4.0×105 g.e. l−1 H2O. The highest concentrations of RV were found in the inflow of the WWTP in September 2007 (2.6×105 g.e. l−1 H2O) and in hospital sewage in September 2007 (4.0×105 g.e. l−1 H2O) (Fig. 1). We furthermore measured the reduction level of NoV in the WWTP at all time-points where NoV was detected in incoming wastewater (n = 15), and observed that NoVs were reduced on average 1.15±0.29SE log10 units in the treatment process.
The majority of all RVs detected belonged to subgroup I
Of the 9 RV-positive samples, 6 (67%) belonged to SGI, and 3 (33%) belonged to SGII. All RV-positive samples found in the hospital wastewater (n = 2) belonged to subgroup SGI; and were collected in September 2007. The two RV subgroups were not simultaneously observed in any samples investigated. We sequenced the VP7 gene of one SGI RV-positive (37-Sept07-Hospital) which had relatively high viral load. This sample was previously negative for the following G-types; G1, G2, G3, G4, G9, G8 and G10, using the nested multiplex PCR assay (data not shown). BLAST analysis of a short 165 nt segment of the VP7-gene revealed high nucleotide homology (99%) to bovine G6 strains isolated in New Zealand (RotaBovG6-NZ), China (CHLY) and India (B-85). Moreover, the nucleotide sequence was also homologous (98% nt similarity) to the human-bovine reassortant vaccine strain (P1 WC3×WI79 [WI79-4]) (Fig. 2A).
(B) Partial capsid NS region (282 bp; 5085 to 5366; ref acc.no. X86557) of NoV GII cDNA (n = 21) isolated from sewage water collected at the local hospital and the WWTP of León, Nicaragua in 2007. Both trees were constructed based on the Kimura two-parameter and neighbor-joining methods by use of MEGA 5.0 software. Bootstrap values are shown at the branch nodes (values of <50% are not shown). RV G4Nic181/05 and Sapporo virus GI/1 (ref. acc. no. U65427) were used as outgroups, respectively. For Nicaraguan strains isolated in this study, the number of the sample and clone is given, followed by month of sample collection and site of collection (HO: Hospital; WWTP: Wastewater Treatment Plant).
We furthermore detected G2 and G4 VP7-specificities, as determined by the nested multiplex PCR assay, in the inflow to the WWTP in July (n = 1) and September 2007 (n = 1), respectively. The VP6 subgroup determination in both samples revealed SGI and SGII specificities, respectively.
The majority of NoVs belonged to genotype GII.4
Of the 37 NoV-positive samples, 28 (76%) were GII strains and 2 (5%) were GI strains, while both NoV genogroups were detected in 7 (19%) of the samples. To study NoV diversity, NoV-positive sewage samples with high viral load were selected for cloning and nucleotide sequencing, and for a subset of these (n = 5) collected in July, September and December of 2007 we obtained clones. In total 21 clones containing a segment (nt position 5085 to 5366) of the NS region of NoV were sequenced, 20 (95%) of these were highly homologous (99% nt similarity) to GII.4 NoV strains. One NoV clone, observed in the inflow of wastewater in September 2007, was highly homologous (100% nt identity) to GII.3 NoV isolated from children with community acquired diarrhea in October 2007 in UK, and to a GII.3 NoV strain isolated from a 30 year old male from Kolkata, India in 2008 (Fig. 2B).
In this study we investigated the prevalence of NoV and RV in hospital and municipal wastewaters in order to understand the transmission of these viruses at community and hospital level following implementation of RV vaccination in Nicaragua.
The major novel finding was the low prevalence of RV, further emphasized by the high prevalence of NoVs in both hospital and municipal wastewater. Several studies have reported higher frequencies of RV as compared to NoV in wastewater. Research groups from China, Tunisia and Germany described detection rates of RV ranging from 32% to 90% and NoV from 3% to 31% , , . In hospital wastewater, Prado and coworkers observed RV in 95% and NoV GII in 28.5% of the samples collected between 2005 and 2008 in Rio de Janeiro, Brazil. . In the current study mean RV prevalence in wastewater from different sources was only 11%, whereas, it was 44% for NoV (p<0.05). Moreover, in hospital wastewater, NoV was detected in 46.4%, whereas RV was only detected in 7.1% of the samples. This suggests a low frequency of severe hospital-requiring RV infections in the community as recently reported . Furthermore, differences were not only observed in virus prevalence, but also in virus concentration; in average RV concentrations were lower as compared to NoV concentrations, both in hospital and in community wastewater. These observations suggest a high NoV transmission at population level in León, Nicaragua, during the time frame investigated, which is supported by the large burden of diarrhea that still persists in León after RV5 introduction .
Taken together, these observations strongly suggest that the implementation of RV5 in the EPI reduced the transmission of RV in León, Nicaragua both at community and hospital level. A recent report further supports this suggestion indicating that the RV immunization program in León is effective in reducing diarrhea episodes during the RV season . In earlier studies from Nicaragua, the dry months (February–April) are usually associated with high rates of RV-induced diarrhea , . In this study, however, most RVs were detected in wastewater during the rainy season (July–September) indicating a shift in the epidemiological pattern for RV. A similar shift has also been observed in a study from United States, reporting a delay of 15 and 8 weeks in the onset and peak of the RV season in 2007 and 2008 respectively, compared to the pre-vaccine periods 2000–2006 . However, the differences in detection rates could also be partially attributed to the study design. Sample collection was not uniform throughout the year; samples were collected weekly at the WWTP during the rainy season and until Christmas holidays (July–December 2007), whereas samples were collected monthly during the remainder of the study period.
The real-time PCR assays used for detection and quantification of these viruses are based on the same chemistry and have similar efficiencies , ; therefore the difference in observed prevalence should not be due to differences in the detection methods, neither due to the concentration method, as ultracentrifugation has been shown to be more efficient to recover RV from wastewater as compared to ultrafiltration-adsorption-elution method .
The NoV concentrations in incoming wastewater for treatment ranged from 1.3×104 to 5.7×106 g.e l−1 H2O. The NoVs were reduced on average 1.15±0.29SE log10 units in the WWTP of León, albeit with variations in reduction between different time points. Furthermore, NoV was found in 32.1% of the outgoing wastewater samples from the treatment plant, demonstrating high transmission of NoV into the water environment even after the treatment process, suggesting that a disinfection step is needed. RV, however, was only detected in one outgoing wastewater sample (3.6%) (Table 1). The low reduction of NoV in the WWTP with subsequent high transmission into the environment is of concern, due to the environmental stability of the virus and its association with food and waterborne outbreaks , .
We characterized RV and NoV by sequencing. The RV concentration in wastewater was low, thus only one partial sequence of the VP7 gene from a water sample where we detected RV SGI-specificity with the real-time PCR assay, designated “37-Sept07-Hospital”, detected in hospital wastewater in September 2007 was obtained. The sequencing data demonstrated relatedness with animal RVs, having a high nucleotide homology (99%) with bovine strains detected in New Zealand, China and India belonging to the G6 genotype, which is the most common genotype found in calves elsewhere , . The “37-Sept07-Hospital” sequence was also highly similar (98% nt homology) with the human-bovine reassortant vaccine strain (P1 WC3×WI79 [WI79-4]), which contains a VP7 protein with G6-specificity of bovine origin . However, the sample containing this bovine-like RV strain was negative for the human G1, G2, G3, G4, G8, G9 and G10 genotypes. The RV5 contains five human-bovine reassortant strains, each with a different human VP7 (G1, G2, G3 or G4) or VP4 (P) specificities that could be detected by RT-PCR . Virus excretion from children vaccinated with RV5 is seldom reported , but can occur . Since this sample was isolated from the hospital wastewater, it is likely that it contains a bovine-like RV from symptomatic children. Previously, a zoonotic bovine RV strain with G8 specificity has been identified in a diarrheic child during the monitoring of RV vaccine program in Nicaragua in 2008 . However, it cannot be ruled out or confirmed that it was the vaccine strain which was detected in this study, as more sequence data will be needed to elucidate this.
The majority (6/9) of RV-positive wastewater samples were of SGI specificity, which is generally associated with human rotavirus G2P . In the current study RV G2 was observed in one wastewater sample, therefore we can speculate but not prove that RV of G2P specificity were circulating in León during the time frame investigated, but at very low frequency. This speculation is strengthened by the fact that G2P represented 88% of the RVs identified in a hospital surveillance carried out in the capital city of Nicaragua (Managua) between 2007 and 2008 . The circulation of RV with P-types not included in the RV5 vaccine, such as P and P might be an expected phenomena after national vaccine implementation . Previous studies from Nicaragua investigating RV genotypes in 2001–2003, 2005 and 2006 , , , found G2P to be the dominating genotype in 2001, but since then circulating at low frequencies. The observed high prevalence of G2P in Nicaragua 2007 and 2008 is likely due to naturally occurring shifts, rather than selection by RV5 introduction, since a very similar re-emergence of G2P strains has been observed in other South- and Central-American countries, where no nationwide vaccine has yet been introduced .
Moreover, it cannot be ruled out that some of the SGI RV strains observed in wastewater were derived from animals, since SGI RVs are more common in animals than in humans , . In the city of León, cattle and other domesticated animals are common and the sewage system also collects dump from these animals.
We observed a low frequency of GI NoVs (5%), and the few samples positive for this genogroup had low concentrations, which made it difficult to further characterize. There are reports from Japan, France and Sweden showing higher frequency of GI NoVs during some months, indicating a high transmission of these viruses in the community , , . NoV GI has often been associated with food and waterborne outbreaks , , . In this study, NoV GII was the most prevalent genogroup throughout the time-period investigated. Samples containing high NoV GII concentrations (>1×105 g.e l−1 H2O) from different sampling and time points were selected for cloning and sequencing. We characterized NoV GII strains from hospital sewage in July, September and December 2007, as well as the inflow to the WWTP in September and December 2007. All NoV clones from the hospital wastewater were genotyped as GII.4 (Fig. 2B). NoV GII.4 has previously been the most common NoV strains detected in pediatric diarrhea in Nicaragua, and are ubiquitous worldwide , . As described elsewhere , the GII.4 Hunter strain was globally replaced by GII.4 2006a/b in early 2006. However, the short segment of the NS region we sequenced in this study is not sufficient to accurately determine the exact GII.4 variant observed. The fact that all clones from the hospital wastewater were genotyped as NoV GII.4 reflects the importance of this genotype in NoV-associated diarrhea in Nicaragua. We found one NoV GII.3 strain in September 2007 in the inflow of the WWTP. The fact that a NoV GII.3 strain was only observed in the municipal wastewater and not in the hospital wastewater might suggest that NoV GII.3 was circulating at community level causing less severe or asymptomatic infections.
The detection of NoV GII.4 strains in July, September and December demonstrates a stable circulation of NoV GII.4 in Nicaragua after RV vaccine introduction. Usually, NoV GII.4 variants are associated with seasonal epidemics and a given variant predominate for a period of time until replaced by a new variant . The presence of one particular NoV variant throughout the year in the current study is interesting, as higher NoV diversity have been observed in water samples from other studies , . However, the differences could be attributed to temporal fluctuations of NoV strains in the community; no clinical data regarding NoV genotypes is available for the same time-period investigated. In an earlier clinical report from Nicaragua, a high diversity of NoV genotypes was found in April to June in children with diarrhea, whereafter GII.4 strains dominated . Unfortunately, in the current study no wastewater samples collected between April and June had sufficient viral concentration to enable NoV genotyping. Whether a change of dynamics of NoV circulation occurs due to the introduction of RV vaccine remains to be elucidated
To conclude, this paper demonstrated low prevalence of RV and high prevalence of NoV in wastewater in León Nicaragua during the post RV vaccination period. The results suggest that implementation of the RV5 vaccine has reduced RV circulating in the community. Determination of RV and NoV in wastewaters may function as a surrogate marker for their prevalence in the society, and thus be used as a tool for monitoring vaccine efficacy.
We thank Dr. Sumit Sharma for critical reading of the manuscript. We further thank Lic. Soledad Calderon, Lic. Margarita Paniagua and Carolin Jönsson for valuable technical assistance; Dr. Felix Espinoza and Luis Escorcia for contact with the office of Water Affairs.
Conceived and designed the experiments: FB PEL LS JN. Performed the experiments: FB JN. Analyzed the data: FB JN. Contributed reagents/materials/analysis tools: PEL LS. Wrote the paper: FB PEL LS JN.
- 1. Patel MM, Widdowson MA, Glass RI, Akazawa K, Vinje J, et al. (2008) Systematic literature review of role of noroviruses in sporadic gastroenteritis. Emerg Infect Dis 14: 1224–1231.MM PatelMA WiddowsonRI GlassK. AkazawaJ. Vinje2008Systematic literature review of role of noroviruses in sporadic gastroenteritis.Emerg Infect Dis1412241231
- 2. Parashar UD, Gibson CJ, Bresse JS, Glass RI (2006) Rotavirus and severe childhood diarrhea. Emerg Infect Dis 12: 304–306.UD ParasharCJ GibsonJS BresseRI Glass2006Rotavirus and severe childhood diarrhea.Emerg Infect Dis12304306
- 3. Parashar UD, Burton A, Lanata C, Boschi-Pinto C, Shibuya K, et al. (2009) Global mortality associated with rotavirus disease among children in 2004. J Infect Dis 200: Suppl 1S9–S15.UD ParasharA. BurtonC. LanataC. Boschi-PintoK. Shibuya2009Global mortality associated with rotavirus disease among children in 2004.J Infect Dis200Suppl 1S9S15
- 4. Iturriza Gomara M, Wong C, Blome S, Desselberger U, Gray J (2002) Molecular characterization of VP6 genes of human rotavirus isolates: correlation of genogroups with subgroups and evidence of independent segregation. J Virol 76: 6596–6601.M. Iturriza GomaraC. WongS. BlomeU. DesselbergerJ. Gray2002Molecular characterization of VP6 genes of human rotavirus isolates: correlation of genogroups with subgroups and evidence of independent segregation.J Virol7665966601
- 5. Nordgren J, Bucardo F, Svensson L, Lindgren PE (2010) Novel light-upon-extension real-time PCR assay for simultaneous detection, quantification, and genogrouping of group A rotavirus. J Clin Microbiol 48: 1859–1865.J. NordgrenF. BucardoL. SvenssonPE Lindgren2010Novel light-upon-extension real-time PCR assay for simultaneous detection, quantification, and genogrouping of group A rotavirus.J Clin Microbiol4818591865
- 6. Duclos P, Okwo-Bele JM, Salisbury D (2011) Establishing global policy recommendations: the role of the Strategic Advisory Group of Experts on immunization. Expert Rev Vaccines 10: 163–173.P. DuclosJM Okwo-BeleD. Salisbury2011Establishing global policy recommendations: the role of the Strategic Advisory Group of Experts on immunization.Expert Rev Vaccines10163173
- 7. Ramani S, Sankaran P, Arumugam R, Sarkar R, Banerjee I, et al. (2010) Comparison of viral load and duration of virus shedding in symptomatic and asymptomatic neonatal rotavirus infections. J Med Virol 82: 1803–1807.S. RamaniP. SankaranR. ArumugamR. SarkarI. Banerjee2010Comparison of viral load and duration of virus shedding in symptomatic and asymptomatic neonatal rotavirus infections.J Med Virol8218031807
- 8. Bucardo F, Nordgren J, Carlsson B, Kindberg E, Paniagua M, et al. (2010) Asymptomatic norovirus infections in Nicaraguan children and its association with viral properties and histo-blood group antigens. Pediatr Infect Dis J 29: 934–939.F. BucardoJ. NordgrenB. CarlssonE. KindbergM. Paniagua2010Asymptomatic norovirus infections in Nicaraguan children and its association with viral properties and histo-blood group antigens.Pediatr Infect Dis J29934939
- 9. Bucardo F, Nordgren J, Carlsson B, Paniagua M, Lindgren PE, et al. (2008) Pediatric norovirus diarrhea in Nicaragua. J Clin Microbiol 46: 2573–2580.F. BucardoJ. NordgrenB. CarlssonM. PaniaguaPE Lindgren2008Pediatric norovirus diarrhea in Nicaragua.J Clin Microbiol4625732580
- 10. Arraj A, Bohatier J, Aumeran C, Bailly JL, Laveran H, et al. (2008) An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis. J Water Health 6: 351–358.A. ArrajJ. BohatierC. AumeranJL BaillyH. Laveran2008An epidemiological study of enteric viruses in sewage with molecular characterization by RT-PCR and sequence analysis.J Water Health6351358
- 11. Barril PA, Giordano MO, Isa MB, Masachessi G, Ferreyra LJ, et al. (2010) Correlation between rotavirus A genotypes detected in hospitalized children and sewage samples in 2006, Cordoba, Argentina. J Med Virol 82: 1277–1281.PA BarrilMO GiordanoMB IsaG. MasachessiLJ Ferreyra2010Correlation between rotavirus A genotypes detected in hospitalized children and sewage samples in 2006, Cordoba, Argentina.J Med Virol8212771281
- 12. Nordgren J, Matussek A, Mattsson A, Svensson L, Lindgren PE (2009) Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant. Water Res 43: 1117–1125.J. NordgrenA. MatussekA. MattssonL. SvenssonPE Lindgren2009Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant.Water Res4311171125
- 13. Myrmel M, Berg EM, Grinde B, Rimstad E (2006) Enteric viruses in inlet and outlet samples from sewage treatment plants. J Water Health 4: 197–209.M. MyrmelEM BergB. GrindeE. Rimstad2006Enteric viruses in inlet and outlet samples from sewage treatment plants.J Water Health4197209
- 14. Kamel AH, Ali MA, El-Nady HG, Aho S, Pothier P, et al. (2010) Evidence of the co-circulation of enteric viruses in sewage and in the population of Greater Cairo. J Appl Microbiol 108: 1620–1629.AH KamelMA AliHG El-NadyS. AhoP. Pothier2010Evidence of the co-circulation of enteric viruses in sewage and in the population of Greater Cairo.J Appl Microbiol10816201629
- 15. Laverick MA, Wyn-Jones AP, Carter MJ (2004) Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage. Lett Appl Microbiol 39: 127–136.MA LaverickAP Wyn-JonesMJ Carter2004Quantitative RT-PCR for the enumeration of noroviruses (Norwalk-like viruses) in water and sewage.Lett Appl Microbiol39127136
- 16. Lodder WJ, de Roda Husman AM (2005) Presence of noroviruses and other enteric viruses in sewage and surface waters in The Netherlands. Appl Environ Microbiol 71: 1453–1461.WJ LodderAM de Roda Husman2005Presence of noroviruses and other enteric viruses in sewage and surface waters in The Netherlands.Appl Environ Microbiol7114531461
- 17. Kremer JR, Langlet J, Skraber S, Weicherding P, Weber B, et al. (2011) Genetic diversity of noroviruses from outbreaks, sporadic cases and wastewater in Luxembourg 2008–2009. Clin Microbiol Infect 17: 1173–1176.JR KremerJ. LangletS. SkraberP. WeicherdingB. Weber2011Genetic diversity of noroviruses from outbreaks, sporadic cases and wastewater in Luxembourg 2008–2009.Clin Microbiol Infect1711731176
- 18. Iwai M, Hasegawa S, Obara M, Nakamura K, Horimoto E, et al. (2009) Continuous presence of noroviruses and sapoviruses in raw sewage reflects infections among inhabitants of Toyama, Japan (2006 to 2008). Appl Environ Microbiol 75: 1264–1270.M. IwaiS. HasegawaM. ObaraK. NakamuraE. Horimoto2009Continuous presence of noroviruses and sapoviruses in raw sewage reflects infections among inhabitants of Toyama, Japan (2006 to 2008).Appl Environ Microbiol7512641270
- 19. Orozco M, Vasquez J, Pedreira C, De Oliveira LH, Amador JJ, et al. (2009) Uptake of rotavirus vaccine and national trends of acute gastroenteritis among children in Nicaragua. J Infect Dis 200: Suppl 1S125–130.M. OrozcoJ. VasquezC. PedreiraLH De OliveiraJJ Amador2009Uptake of rotavirus vaccine and national trends of acute gastroenteritis among children in Nicaragua.J Infect Dis200Suppl 1S125130
- 20. Becker-Dreps S, Paniagua M, Dominik R, Cao H, Shah NK, et al. (2011) Changes in childhood diarrhea incidence in nicaragua following 3 years of universal infant rotavirus immunization. Pediatr Infect Dis J 30: 243–247.S. Becker-DrepsM. PaniaguaR. DominikH. CaoNK Shah2011Changes in childhood diarrhea incidence in nicaragua following 3 years of universal infant rotavirus immunization.Pediatr Infect Dis J30243247
- 21. Bucardo F, Karlsson B, Nordgren J, Paniagua M, Gonzalez A, et al. (2007) Mutated G4P rotavirus associated with a nationwide outbreak of gastroenteritis in Nicaragua in 2005. J Clin Microbiol 45: 990–997.F. BucardoB. KarlssonJ. NordgrenM. PaniaguaA. Gonzalez2007Mutated G4P rotavirus associated with a nationwide outbreak of gastroenteritis in Nicaragua in 2005.J Clin Microbiol45990997
- 22. Nordgren J, Bucardo F, Dienus O, Svensson L, Lindgren PE (2008) Novel light-upon-extension real-time PCR assays for detection and quantification of genogroup I and II noroviruses in clinical specimens. J Clin Microbiol 46: 164–170.J. NordgrenF. BucardoO. DienusL. SvenssonPE Lindgren2008Novel light-upon-extension real-time PCR assays for detection and quantification of genogroup I and II noroviruses in clinical specimens.J Clin Microbiol46164170
- 23. Swedish Environmental Protection Agency (2008) Guidance to the Swedish Environmental Protection Agency's regulation for environmental reports (Vägledning om Naturvårdsverkets föreskrifter om miljörapport).Swedish Environmental Protection Agency2008Guidance to the Swedish Environmental Protection Agency's regulation for environmental reports (Vägledning om Naturvårdsverkets föreskrifter om miljörapport).
- 24. Iturriza-Gomara M, Kang G, Gray J (2004) Rotavirus genotyping: keeping up with an evolving population of human rotaviruses. J Clin Virol 31: 259–265.M. Iturriza-GomaraG. KangJ. Gray2004Rotavirus genotyping: keeping up with an evolving population of human rotaviruses.J Clin Virol31259265
- 25. Kojima S, Kageyama T, Fukushi S, Hoshino FB, Shinohara M, et al. (2002) Genogroup-specific PCR primers for detection of Norwalk-like viruses. J Virol Methods 100: 107–114.S. KojimaT. KageyamaS. FukushiFB HoshinoM. Shinohara2002Genogroup-specific PCR primers for detection of Norwalk-like viruses.J Virol Methods100107114
- 26. He XQ, Cheng L, Zhang DY, Xie XM, Wang DH, et al. (2011) One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment. Water Sci Technol 63: 191–198.XQ HeL. ChengDY ZhangXM XieDH Wang2011One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment.Water Sci Technol63191198
- 27. Sdiri-Loulizi K, Hassine M, Aouni Z, Gharbi-Khelifi H, Chouchane S, et al. (2010) Detection and molecular characterization of enteric viruses in environmental samples in Monastir, Tunisia between January 2003 and April 2007. J Appl Microbiol 109: 1093–1104.K. Sdiri-LouliziM. HassineZ. AouniH. Gharbi-KhelifiS. Chouchane2010Detection and molecular characterization of enteric viruses in environmental samples in Monastir, Tunisia between January 2003 and April 2007.J Appl Microbiol10910931104
- 28. Hamza IA, Jurzik L, Stang A, Sure K, Uberla K, et al. (2009) Detection of human viruses in rivers of a densly-populated area in Germany using a virus adsorption elution method optimized for PCR analyses. Water Res 43: 2657–2668.IA HamzaL. JurzikA. StangK. SureK. Uberla2009Detection of human viruses in rivers of a densly-populated area in Germany using a virus adsorption elution method optimized for PCR analyses.Water Res4326572668
- 29. Prado T, Silva DM, Guilayn WC, Rose TL, Gaspar AM, et al. (2011) Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants. Water Res 45: 1287–1297.T. PradoDM SilvaWC GuilaynTL RoseAM Gaspar2011Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants.Water Res4512871297
- 30. Mast TC, Khawaja S, Espinoza F, Paniagua M, Palacio Del Carmen L, et al. (2011) Case-control Study of the Effectiveness of Vaccination With Pentavalent Rotavirus Vaccine in Nicaragua. Pediatr Infect Dis J 30: TC MastS. KhawajaF. EspinozaM. PaniaguaL. Palacio Del Carmen2011Case-control Study of the Effectiveness of Vaccination With Pentavalent Rotavirus Vaccine in Nicaragua.Pediatr Infect Dis J30
- 31. Espinoza F, Bucardo F, Paniagua M, Svensson L, Hallander HO, et al. (2006) Shifts of rotavirus g and p types in Nicaragua–2001–2003. Pediatr Infect Dis J 25: 1078–1080.F. EspinozaF. BucardoM. PaniaguaL. SvenssonHO Hallander2006Shifts of rotavirus g and p types in Nicaragua–2001–2003.Pediatr Infect Dis J2510781080
- 32. Tate JE, Panozzo CA, Payne DC, Patel MM, Cortese MM, et al. (2009) Decline and change in seasonality of US rotavirus activity after the introduction of rotavirus vaccine. Pediatrics 124: 465–471.JE TateCA PanozzoDC PayneMM PatelMM Cortese2009Decline and change in seasonality of US rotavirus activity after the introduction of rotavirus vaccine.Pediatrics124465471
- 33. Fumian TM, Leite JP, Castello AA, Gaggero A, Caillou MS, et al. (2010) Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration. J Virol Methods 170: 42–46.TM FumianJP LeiteAA CastelloA. GaggeroMS Caillou2010Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration.J Virol Methods1704246
- 34. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, et al. (1999) Food-related illness and death in the United States. Emerg Infect Dis 5: 607–625.PS MeadL. SlutskerV. DietzLF McCaigJS Bresee1999Food-related illness and death in the United States.Emerg Infect Dis5607625
- 35. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, et al. (2002) Waterborne outbreak of Norwalk-like virus gastroenteritis at a tourist resort, Italy. Emerg Infect Dis 8: 563–568.D. BocciaAE TozziB. CotterC. RizzoT. Russo2002Waterborne outbreak of Norwalk-like virus gastroenteritis at a tourist resort, Italy.Emerg Infect Dis8563568
- 36. Cashman O, Lennon G, Sleator RD, Power E, Fanning S, et al. (2010) Changing profile of the bovine rotavirus G6 population in the south of Ireland from 2002 to 2009. Vet Microbiol 146: 238–244.O. CashmanG. LennonRD SleatorE. PowerS. Fanning2010Changing profile of the bovine rotavirus G6 population in the south of Ireland from 2002 to 2009.Vet Microbiol146238244
- 37. Alkan F, Ozkul A, Oguzoglu TC, Timurkan MO, Caliskan E, et al. (2010) Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Turkish calves with diarrhea, 1997–2008. Vet Microbiol 141: 231–237.F. AlkanA. OzkulTC OguzogluMO TimurkanE. Caliskan2010Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Turkish calves with diarrhea, 1997–2008.Vet Microbiol141231237
- 38. Matthijnssens J, Joelsson DB, Warakomski DJ, Zhou T, Mathis PK, et al. (2010) Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq. Virology 403: 111–127.J. MatthijnssensDB JoelssonDJ WarakomskiT. ZhouPK Mathis2010Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq.Virology403111127
- 39. Payne DC, Edwards KM, Bowen MD, Keckley E, Peters J, et al. (2010) Sibling transmission of vaccine-derived rotavirus (RotaTeq) associated with rotavirus gastroenteritis. Pediatrics 125: e438–441.DC PayneKM EdwardsMD BowenE. KeckleyJ. Peters2010Sibling transmission of vaccine-derived rotavirus (RotaTeq) associated with rotavirus gastroenteritis.Pediatrics125e438441
- 40. Vesikari T, Matson DO, Dennehy P, Van Damme P, Santosham M, et al. (2006) Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 354: 23–33.T. VesikariDO MatsonP. DennehyP. Van DammeM. Santosham2006Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine.N Engl J Med3542333
- 41. Yen C, Jakob K, Esona MD, Peckham X, Rausch J, et al. (2011) Detection of fecal shedding of rotavirus vaccine in infants following their first dose of pentavalent rotavirus vaccine. Vaccine 29: 4151–4155.C. YenK. JakobMD EsonaX. PeckhamJ. Rausch2011Detection of fecal shedding of rotavirus vaccine in infants following their first dose of pentavalent rotavirus vaccine.Vaccine2941514155
- 42. Banyai K, Esona MD, Mijatovic S, Kerin TK, Pedreira C, et al. (2009) Zoonotic bovine rotavirus strain in a diarrheic child, Nicaragua. J Clin Virol 46: 391–393.K. BanyaiMD EsonaS. MijatovicTK KerinC. Pedreira2009Zoonotic bovine rotavirus strain in a diarrheic child, Nicaragua.J Clin Virol46391393
- 43. Patel M, Pedreira C, De Oliveira LH, Tate J, Orozco M, et al. (2009) Association between pentavalent rotavirus vaccine and severe rotavirus diarrhea among children in Nicaragua. JAMA 301: 2243–2251.M. PatelC. PedreiraLH De OliveiraJ. TateM. Orozco2009Association between pentavalent rotavirus vaccine and severe rotavirus diarrhea among children in Nicaragua.JAMA30122432251
- 44. Bourdett-Stanziola L, Ortega-Barria E, Espinoza F, Bucardo F, Jimenez C, et al. (2010) Rotavirus genotypes in Costa Rica, Nicaragua, Honduras and the Dominican Republic. Intervirology 53: 390–393.L. Bourdett-StanziolaE. Ortega-BarriaF. EspinozaF. BucardoC. Jimenez2010Rotavirus genotypes in Costa Rica, Nicaragua, Honduras and the Dominican Republic.Intervirology53390393
- 45. Matthijnssens J, Bilcke J, Ciarlet M, Martella V, Banyai K, et al. (2009) Rotavirus disease and vaccination: impact on genotype diversity. Future Microbiol 4: 1303–1316.J. MatthijnssensJ. BilckeM. CiarletV. MartellaK. Banyai2009Rotavirus disease and vaccination: impact on genotype diversity.Future Microbiol413031316
- 46. Tang B, Gilbert JM, Matsui SM, Greenberg HB (1997) Comparison of the rotavirus gene 6 from different species by sequence analysis and localization of subgroup-specific epitopes using site-directed mutagenesis. Virology 237: 89–96.B. TangJM GilbertSM MatsuiHB Greenberg1997Comparison of the rotavirus gene 6 from different species by sequence analysis and localization of subgroup-specific epitopes using site-directed mutagenesis.Virology2378996
- 47. Kitajima M, Oka T, Haramoto E, Takeda N, Katayama K, et al. (2010) Seasonal distribution and genetic diversity of genogroups I, II, and IV noroviruses in the Tamagawa River, Japan. Environ Sci Technol 44: 7116–7122.M. KitajimaT. OkaE. HaramotoN. TakedaK. Katayama2010Seasonal distribution and genetic diversity of genogroups I, II, and IV noroviruses in the Tamagawa River, Japan.Environ Sci Technol4471167122
- 48. da Silva AK, Le Saux JC, Parnaudeau S, Pommepuy M, Elimelech M, et al. (2007) Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II. Appl Environ Microbiol 73: 7891–7897.AK da SilvaJC Le SauxS. ParnaudeauM. PommepuyM. Elimelech2007Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II.Appl Environ Microbiol7378917897
- 49. Nordgren J, Kindberg E, Lindgren PE, Matussek A, Svensson L (2010) Norovirus gastroenteritis outbreak with a secretor-independent susceptibility pattern, Sweden. Emerg Infect Dis 16: 81–87.J. NordgrenE. KindbergPE LindgrenA. MatussekL. Svensson2010Norovirus gastroenteritis outbreak with a secretor-independent susceptibility pattern, Sweden.Emerg Infect Dis168187
- 50. Lysen M, Thorhagen M, Brytting M, Hjertqvist M, Andersson Y, et al. (2009) Genetic diversity among food-borne and waterborne norovirus strains causing outbreaks in Sweden. J Clin Microbiol 47: 2411–2418.M. LysenM. ThorhagenM. BryttingM. HjertqvistY. Andersson2009Genetic diversity among food-borne and waterborne norovirus strains causing outbreaks in Sweden.J Clin Microbiol4724112418
- 51. CDC (2007) Norovirus outbreak associated with ill food-service workers–Michigan, January–February 2006. MMWR Morbidity and mortality weekly report 56: 1212–1216.CDC2007Norovirus outbreak associated with ill food-service workers–Michigan, January–February 2006.MMWR Morbidity and mortality weekly report5612121216
- 52. Siebenga JJ, Vennema H, Zheng DP, Vinje J, Lee BE, et al. (2009) Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001–2007. J Infect Dis 200: 802–812.JJ SiebengaH. VennemaDP ZhengJ. VinjeBE Lee2009Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001–2007.J Infect Dis200802812
- 53. Gallimore CI, Iturriza-Gomara M, Xerry J, Adigwe J, Gray JJ (2007) Inter-seasonal diversity of norovirus genotypes: emergence and selection of virus variants. Arch Virol 152: 1295–1303.CI GallimoreM. Iturriza-GomaraJ. XerryJ. AdigweJJ Gray2007Inter-seasonal diversity of norovirus genotypes: emergence and selection of virus variants.Arch Virol15212951303
- 54. Aw TG, Gin KY, Ean Oon LL, Chen EX, Woo CH (2009) Prevalence and genotypes of human noroviruses in tropical urban surface waters and clinical samples in Singapore. Appl Environ Microbiol 75: 4984–4992.TG AwKY GinLL Ean OonEX ChenCH Woo2009Prevalence and genotypes of human noroviruses in tropical urban surface waters and clinical samples in Singapore.Appl Environ Microbiol7549844992