Molecular Epidemiology of Norovirus in Outbreaks of Gastroenteritis in Southwest Germany from 2001 to 2004
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微生物临床杂志 2006年第4期
Institute for Environmental and Animal Hygiene, Hohenheim University, Stuttgart, Germany
Baden-Württemberg State Health Office District Government Stuttgart, Stuttgart, Germany
Stuttgart Regional Chemical and Veterinary Control Laboratories, Fellbach, Germany
ABSTRACT
The identification and molecular epidemiology of norovirus in outbreaks of gastroenteritis were studied during a 3-year period in Germany. Specimens (n = 316) from 159 nonbacterial gastroenteritis outbreaks from March 2001 to June 2004 were analyzed for the presence of noroviruses by reverse transcriptase PCR. Outbreaks were most frequent in elderly people's homes and care centers (43%), followed by hospitals (24%). Molecular analyses of strains from 148 outbreaks showed that there were up to 12 genotypes involved in the outbreaks. Genogroup II noroviruses were responsible for 95% of the outbreaks. Cocirculation of more than one strain in the same outbreak and cocirculation of genogroup I and II strains in the same place were observed. Genogroup II4 (Grimsby-like) was the most prevalent strain, accounting for 48% and 67% of the outbreaks in 2002 and 2003, respectively. The genogroup IIb (Castell/Suria) genotype was observed in all the years of the study. Epidemiological and molecular data indicated that there was a major shift of the predominant strain that coincided with the appearance of a new variant of genogroup II4 in 2002. By the application of reverse transcriptase PCR, this study has demonstrated the importance and dynamism of noroviruses in Germany.
INTRODUCTION
Noroviruses (NV) are single-stranded RNA viruses belonging to the family Caliciviridae. NV are recognized as an important cause of acute nonbacterial outbreaks of gastroenteritis (11, 15, 16, 33). Outbreaks of NV affect people of all age groups (11) and have been reported to occur in a variety of settings, especially in semiclosed communities such as families (27), schools (7), elderly people's homes (33), hospitals (35), hotels (27), and cruise ships (1, 8). Outbreaks involving a large number of individuals are usually transmitted through common sources such as food and water (4, 10), while transmission to other uninfected individuals during outbreaks is usually by person-to-person contact (10). The viruses have also been identified in sporadic cases of gastroenteritis (13). While most of the outbreaks are known to have a seasonal pattern (32), sporadic cases of disease usually occur throughout the year (27).
Noroviruses are one of four genera of the Caliciviridae and are divided into genogroup I (GGI) and genogroup II (GGII), based on genetic diversity (2, 22). Molecular characterization is an important tool in understanding the pattern and distribution of outbreaks caused by this group of agents. Genomic analysis reveals a wide diversity within this genus, even in the RNA-dependent RNA polymerase (RdRp) gene, which is considered to be conserved (2, 9). The lower diversity of the RdRp gene has led to the adoption of this region as a target for the diagnosis of gastroenteritis caused by these agents (3, 28), as well as for molecular and epidemiological studies of calicivirus infections (16, 18).
Based on sequence diversity, NV from the two genogroups have been divided into 17 clusters, with 5 in GGI, 11 in GGII, and 1 cluster equidistant from the two genogroups (37). The prototype virus for the genus is the Norwalk virus (GGI/1), which belongs to GGI, with other members of GGI, including Southampton (GGI/2), Desert Shield (GGI/3), Chiba (GGI/4), and Musgrove (GGI/5). The GGII viruses include Hawaii (GGII/1), Melksham (GGII/2), Toronto (GGII/3), Lordsdale (GGII/4), Hillingdon (GGII/5), Florida (GGII/6), and Leads (GGII/7), while Alphatron (GGIV.1) clusters between the two genogroups (33, 37). Strains are identified as belonging to the same clusters (genotype) if they code for more than 80% amino acid sequence identity in the complete capsid gene or if they code for more than 85% or 90% amino acid sequence identity for GGI and -II, respectively, in the RdRp gene (36).
The objectives of this study were to determine the prevalence of NV in stool samples from outbreaks of acute gastroenteritis in Baden-Württemberg, Germany, from March 2001 to June 2004 and to characterize the types and distribution of the viruses identified in the outbreaks. This will help in the understanding of the significance and epidemiology of the virus in and beyond the studied area.
MATERIALS AND METHODS
Stool samples. Over a 4-year period from 2001 to June 2004, 544 outbreaks were reported to the Baden-Württemberg State Health Office, Germany. Submitted stool samples were tested for bacterial and viral pathogens, including NV. Out of these 544 outbreaks, 244 (44.9%) were positive for NV. One hundred fifty-nine outbreaks (approximately 1,300 samples) were selected randomly from the individual years in proportion to the number of outbreaks that were positive in that year. The overall number of infected persons was 3,499, with a range of 5 to 150. In total, 316 samples from the 159 outbreaks were selected and sequenced (usually two samples per outbreak). The sequenced samples consisted of 13 outbreaks (26 samples) from 2001, 99 outbreaks (196 samples) from 2002, 35 outbreaks (70 sequences) from 2003, and 12 outbreaks (24 sequences) from 2004. NV testing was done by reverse transcriptase PCR (RT-PCR).
RNA extraction. Viral RNA was extracted from 300 μl of 20% stool suspensions by using size-fractionated silica particles (Sigma-Aldrich Steinheim, Germany) to bind the RNA in the presence of guanidinium isothiocynate (6). After being washed in a guanidinium-containing buffer, the RNA was eluted in nuclease-free water containing 40 U RNase inhibitor (RNasin; Promega, Madison, Wis.) and was either reverse transcribed immediately or stored at –70°C.
RT-PCR. A nested RT-PCR method was used for testing RNA from stool samples for NV, using the primer pair NV32 (5'-4226-4245ATG AAT ATG AAT GAA GAT GG-3') and NV36 (5'-4707-4726ATT GGT CCT TCT GTT TTG TC-3') for first-round amplification and primer pair NV33 (5'-4280-4299TAC CAC TAT GAT GCA GAT TA-3') and NV35 (5'-4617-4636GTT GAC ACA ATC TCA TCA TC-3') for second-round amplification (numbers refer to the nucleotide positions of Lordsdale virus [GenBank accession no. X86557] [33]). The final product is a 338-bp portion of the RdRp gene of both genogroups I and II. Briefly, 5 μl of the extracted RNA was added to a 15-μl mix containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, each deoxynucleoside triphosphate (Amersham Pharmacia Biotech, Piscataway, NJ) at a concentration of 1 mM, and 20 U of avian myeloblastoma virus reverse transcriptase (Roche Applied Science, Mannheim, Germany). Reverse transcription was performed at 25°C for 10 min, followed by 42°C for 60 min, at the end of which the reaction was stopped by denaturing the enzyme at 98°C for 5 min and the mixture was cooled down to 4°C. Two microliters of the resulting cDNA was added to 48 μl of the PCR mix in a 50-μl reaction mixture consisting of 10 mM Tris-HCl, 50 mM KCl, 1.25 mM MgCl2, 2.5 U of Taq polymerase (Promega, Madison, Wis.), each deoxynucleoside triphosphate at a concentration of 125 μM, and 30 pmol each of the NV32 and NV36 primers. Amplification was performed with the following cycling profile: initial denaturation at 94°C for 60 s; 35 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, and extension at 72°C for 45 s; and a final extension at 72°C for 3 min. The nested PCR was performed using 1 μl of the first PCR product under the same reaction conditions with primers NV33 and NV35. The final product was examined by gel electrophoresis in 2% agarose gels containing ethidium bromide.
Nucleotide sequencing. The 338-bp product from the RT-PCR was purified with the Qiaquick kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. For DNA sequencing reaction, the fluorescence-labeled dideoxynucleotide technology from Applied Biosystems was used. The sequenced fragments were separated, and the data were collected with an automated sequencer (model 310; Applied Biosystems, Foster City, CA).
Nucleotide sequence analysis. Sequences were aligned with the ClustalW program (34). Phylogenetic analyses were done with the PHYLIP 3.6 package (12), using the Jukes-Cantor distance and neighbor-joining methods. Statistical confidence for the evolutionary trees was assessed by bootstrap analysis (1,000 replicates) and trees drawn with the Treeview program (31). The sample numbers are presented in the phylogenetic tree, preceded by the year of the outbreak and the outbreak number. The Manchester strain of sapoviruses (25) was used as the outgroup; also included are 14 reference strains taken from GenBank and 8 previously published and unpublished strains, also from GenBank (7, 17, 19). The Rostock039 virus (accession no. AF312520), identified in Germany in 2000, was used to represent the Grimsby-like sequences.
Nucleotide sequence accession numbers. The nucleotide sequence data have been submitted to GenBank and assigned accession numbers DQ157056 to DQ157140.
RESULTS
Outbreaks occurred in all parts of Baden-Württemberg and in many different settings, which can be grouped as shown in Table 1. Most of the outbreaks took place in medical facilities (66.7%; n = 106 outbreaks), followed by restaurants and catering facilities (17%; n = 27 outbreaks), facilities for children and teenagers (11.9%; n = 19 outbreaks), and private celebrations (3.1%; n = 5 outbreaks).
The distribution of the outbreaks within the study period shows that outbreaks occurred in all the months of 2002 but not in all months in other years (Fig. 1). Furthermore, there were also seasonal differences observed; 72% of all outbreaks occurred in autumn and winter, from October to March.
Ninety-nine (62.3%; n = 159) of the outbreaks took place in 2002. In this year, 35 outbreaks occurred in December alone. Most of the outbreaks in 2003 took place at the beginning of the year, with 13 outbreaks in the month of January alone. These could be seen as a part of the epidemic that started in the previous year.
To determine the genetic diversity of the noroviruses studied, 282-nucleotide stretches from the polymerase regions of 316 samples obtained from 159 outbreaks were sequenced. Pairwise alignment of two sequences from each outbreak showed that the sequences of noroviruses from 119 outbreaks (74.8%) were exactly the same, noroviruses from 13 outbreaks (8.1%) had one or two nucleotide differences, those from six outbreaks (3.8%) had more than two nucleotide differences despite repetition of sequencing, those from nine outbreaks (5.7%) could not be compared (only one sample from each of these outbreaks was successfully sequenced), and those from eight outbreaks (5%) had nonreproducible results (that is, one or both sequences were different on repeated sequencing). Although this phenomenon has already been reported in other studies (21), the results were regarded as inconclusive. Furthermore, there were four outbreaks from which only one sample per outbreak was obtained. Out of the 316 samples sequenced, 265 sequences, including 25 sequences from 2001, 153 sequences from 2002, 63 sequences from 2003, and 24 sequences from 2004, were used to generate a phylogenetic tree (Fig. 2). In all, sequences from 148 outbreaks were used for the alignment.
The sequence analysis shows that genogroup II was predominant in all of the years, accounting for 95.3% of the outbreaks used for the phylogenetic tree and 9 of the 12 clusters in which a sequence(s) from this study was included. There were seven clusters in 2001, eight clusters in 2002, six in 2003, and four in 2004 when the phylogenetic trees for the individual years were reconstructed (results not shown), indicating a reduction in the number of circulating strains in 2003 and 2004. All of the sequences from the same outbreaks in 2001 and 2004 clustered together. However, the sequences from one outbreak (2.9%) in 2003 and two outbreaks (2.1%) in 2002 separated into different clusters, corresponding to the results of the pairwise alignment. This is despite the fact that only sequences of outbreaks in which there were fewer than three base differences after repeated sequencing were included in the phylogenetic analysis.
Forty-four sequences from 25 outbreaks clustered with the Suria and Castell viruses, which were identified by Buesa et al. (7) and designated GGIIb. A predominant number of the outbreaks in 2001 (four outbreaks) clustered with Melksham virus and the Castell/Suria viruses. For the rest of the years, most of the outbreaks clustered with Camberwell virus (GGII/4). This was most obvious for 2002 and 2003, in which 102 (66.7%) and 47 (74.6%) of all the sequences in the phylogenetic tree belonged to this cluster, respectively. Out of the Camberwell cluster, 84 (82.4%) strains in 2002 and 43 (91.5%) strains in 2003 were found to have the unusual mutation of AACTTG to AATCTG at position 4820 of Norwalk virus (accession no. M87661) when related to the Grimsby and Grimsby-like viruses (26). "New variant" was used for the viruses identified with this mutation to differentiate them from the closely related Grimsby and Grimsby-like viruses. In all, 133 sequences forming 77.7% of the Camberwell cluster and 50.1% of all the sequences analyzed belonged to the variant. These groups of viruses were found at a lower percentage (25%) in 2004 and not found among all the sequences analyzed in 2001.
The 2001 and 2002 GGI strains did not completely resolve into any of the reference strains. The 2001 strain had a bootstrap value of 270 with Southampton virus (accession no. L07418), its closest reference strain, while the 2002 strain had a bootstrap value of 419 with BS5 virus (accession no. AF093797), its closest reference strain. However, the 2003 and 2004 strains clustered with the Southampton (bootstrap value, 1,000) and Norwalk (bootstrap value, 994) viruses, respectively (results not shown). In the general tree (Fig. 2), the unresolved 2001 and 2002 strains clustered together, with a bootstrap value of 365 with BS5 virus, their common closest reference strain, while for 2003 and 2004, all strains clustered the same as in the individual trees. The GGI clusters had a similarity of 92 to 100% for nucleotide sequences within individual groups, while the GGII clusters had a similarity of 78 to 100% for nucleotide sequences within the groups for the analyzed region. Among all the clusters, the nucleotide sequence similarities were 74 to 100% for GGI, 60 to 100% for GGII, and 49 to 67% between the genogroups (Table 2).
Some virus strains were found to have been involved in more than one outbreak in the same area, and circulation of more than one strain at the same place in the same year was also seen. There was also cocirculation of more than one genotype at the same period, as outbreaks that were reported in the same month and period were found in different clusters.
DISCUSSION
Samples from outbreaks of acute gastroenteritis from the state of Baden-Württemberg are sent to the State Health Office in Stuttgart on a regular basis. Our results show that NV account for 44.9% of all outbreaks of gastroenteritis reported during the study period. A representative number of these outbreaks were selected for further study of their epidemiological characteristics.
Our results showed that medical facilities accounted for 66.7% of the outbreaks and 70.2% of the persons infected in this study. This was followed by outbreaks in food outlets (restaurants and catering facilities), which accounted for 17% of the outbreaks. In an earlier study in Germany, all settings making up medical facilities together accounted for over 90% of all outbreaks studied (32), while in the United Kingdom, hospitals as the only medical facility accounted for 86.7% of all outbreaks studied (27). Both values are higher than the 66.7% we recorded; however, our value was higher than the 43% share of outbreaks in medical facilities reported in the United States (11).
As seen from the phylogenetic tree, the GGII strains of the virus were found to be responsible for most (95.3%) of the outbreaks. Studies from different parts of the world have also reported the predominance of GGII, e.g., in France (5), The Netherlands (24), the United Kingdom (27), Australia (23), Canada (16), and the United States (11, 29), with many of them in the same range as our value. Among the GGI strains identified in this study, two outbreaks (outbreak 151 in 2001 and outbreak 007 in 2002) clustered together but failed to completely resolve into any of the reference strains used. This may be attributed to the circulation of a new strain or to the position of the genome used in the study. While the RNA polymerase region is mostly used for diagnosis of NV (3), a complete identification requires the use of the capsid region (21).
The identification of many different virus strains, belonging to 12 clusters within the 40 months of this study, shows that many genotypes were circulating in the region during this period. An increase in the number of clusters was observed in 2002 compared to 2001. Thereafter, there was a gradual decrease in the number of clusters circulating over the years. The minimum percent similarity within the Camberwell virus group (78%), which is below the 90% expected for GGII within the polymerase region (36), indicates that the group may contain more than one genotype.
In this study we found the involvement of more than one strain of virus in the same outbreak, indicating that two different genotypes may have been involved. This was further confirmed by sequencing more amplicons from three selected outbreaks with this pattern (results not shown). There were also cases where two different sequences were obtained from the same stool sample, but the results were inconclusive and the sequences were not included in the analysis. The existence of multiple sequences in an outbreak is usually associated with shellfish-related outbreaks due to the ability of shellfish to concentrate NV from a contaminated environment as a result of their filter-feeding mechanism (20). However, some other studies have reported the presence of multiple sequences in outbreaks not related to shellfish and even in sporadic cases (7, 21). The circulation of more than one strain in the same place raises the possibility of more than one virus strain being involved in the same outbreak.
The GGII4 "new variant" (26), which clustered with the Camberwell virus (Fig. 2), first emerged in 2002 and became the predominant circulating strain in the same year. The same variant of virus was also identified as the predominant strain in other European countries (26). Incidentally, Rostock039, isolated in Germany in 2000 (accession no. AF312520) (unpublished report), had the Grimsby-like earlier sequence of AACTTG. Although this group of viruses is said to have been responsible for the epidemic outbreak of acute gastroenteritis that was observed in 2002 and spilled over into the early part of 2003, it does not appear as if it will remain endemic as reported for Grimsby virus in the United Kingdom (15). Not only did the number of outbreaks attributed to this virus start going down in 2003, but the percentage of outbreaks caused by the group fell dramatically from 68.3% in 2003 to 25% in 2004.
The Castell and Suria strain group, which has been reported in various parts of Europe and was also reported in drinking water in Sweden (30), was also found in all the years of this study. This group and the Melksham virus group were the predominant strains in 2001. The Castell/Suria strains were reported to be related to Mexico virus by capsid analysis (7). Mexico virus was part of the group identified to have been replaced at the beginning of 2002 by the new GGII4 variant in Europe (26).
In this study, we have been able to demonstrate clearly that NV are an important cause of gastroenteritis in Germany. We showed that there are more than three genotypes involved in causing outbreaks of acute gastroenteritis throughout the period of this study. More work needs to be done to characterize the virus strains identified in this study fully and to understand the involvement of multiple sequences in individual infections and outbreaks. The results of this work will contribute to a better understanding of the epidemiology and pathogenicity of NV in Germany.
ACKNOWLEDGMENTS
We are grateful to all the staff of the molecular laboratory of the State Health Office Stuttgart and particularly to Christine Herrmann and Elke Gutwein for some of the laboratory work.
This study was partially supported by the Landesstiftung Baden-Württenberg gGmbH, Stuttgart, Germany (grant P-LS-E/MOP A8-8).
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Baden-Württemberg State Health Office District Government Stuttgart, Stuttgart, Germany
Stuttgart Regional Chemical and Veterinary Control Laboratories, Fellbach, Germany
ABSTRACT
The identification and molecular epidemiology of norovirus in outbreaks of gastroenteritis were studied during a 3-year period in Germany. Specimens (n = 316) from 159 nonbacterial gastroenteritis outbreaks from March 2001 to June 2004 were analyzed for the presence of noroviruses by reverse transcriptase PCR. Outbreaks were most frequent in elderly people's homes and care centers (43%), followed by hospitals (24%). Molecular analyses of strains from 148 outbreaks showed that there were up to 12 genotypes involved in the outbreaks. Genogroup II noroviruses were responsible for 95% of the outbreaks. Cocirculation of more than one strain in the same outbreak and cocirculation of genogroup I and II strains in the same place were observed. Genogroup II4 (Grimsby-like) was the most prevalent strain, accounting for 48% and 67% of the outbreaks in 2002 and 2003, respectively. The genogroup IIb (Castell/Suria) genotype was observed in all the years of the study. Epidemiological and molecular data indicated that there was a major shift of the predominant strain that coincided with the appearance of a new variant of genogroup II4 in 2002. By the application of reverse transcriptase PCR, this study has demonstrated the importance and dynamism of noroviruses in Germany.
INTRODUCTION
Noroviruses (NV) are single-stranded RNA viruses belonging to the family Caliciviridae. NV are recognized as an important cause of acute nonbacterial outbreaks of gastroenteritis (11, 15, 16, 33). Outbreaks of NV affect people of all age groups (11) and have been reported to occur in a variety of settings, especially in semiclosed communities such as families (27), schools (7), elderly people's homes (33), hospitals (35), hotels (27), and cruise ships (1, 8). Outbreaks involving a large number of individuals are usually transmitted through common sources such as food and water (4, 10), while transmission to other uninfected individuals during outbreaks is usually by person-to-person contact (10). The viruses have also been identified in sporadic cases of gastroenteritis (13). While most of the outbreaks are known to have a seasonal pattern (32), sporadic cases of disease usually occur throughout the year (27).
Noroviruses are one of four genera of the Caliciviridae and are divided into genogroup I (GGI) and genogroup II (GGII), based on genetic diversity (2, 22). Molecular characterization is an important tool in understanding the pattern and distribution of outbreaks caused by this group of agents. Genomic analysis reveals a wide diversity within this genus, even in the RNA-dependent RNA polymerase (RdRp) gene, which is considered to be conserved (2, 9). The lower diversity of the RdRp gene has led to the adoption of this region as a target for the diagnosis of gastroenteritis caused by these agents (3, 28), as well as for molecular and epidemiological studies of calicivirus infections (16, 18).
Based on sequence diversity, NV from the two genogroups have been divided into 17 clusters, with 5 in GGI, 11 in GGII, and 1 cluster equidistant from the two genogroups (37). The prototype virus for the genus is the Norwalk virus (GGI/1), which belongs to GGI, with other members of GGI, including Southampton (GGI/2), Desert Shield (GGI/3), Chiba (GGI/4), and Musgrove (GGI/5). The GGII viruses include Hawaii (GGII/1), Melksham (GGII/2), Toronto (GGII/3), Lordsdale (GGII/4), Hillingdon (GGII/5), Florida (GGII/6), and Leads (GGII/7), while Alphatron (GGIV.1) clusters between the two genogroups (33, 37). Strains are identified as belonging to the same clusters (genotype) if they code for more than 80% amino acid sequence identity in the complete capsid gene or if they code for more than 85% or 90% amino acid sequence identity for GGI and -II, respectively, in the RdRp gene (36).
The objectives of this study were to determine the prevalence of NV in stool samples from outbreaks of acute gastroenteritis in Baden-Württemberg, Germany, from March 2001 to June 2004 and to characterize the types and distribution of the viruses identified in the outbreaks. This will help in the understanding of the significance and epidemiology of the virus in and beyond the studied area.
MATERIALS AND METHODS
Stool samples. Over a 4-year period from 2001 to June 2004, 544 outbreaks were reported to the Baden-Württemberg State Health Office, Germany. Submitted stool samples were tested for bacterial and viral pathogens, including NV. Out of these 544 outbreaks, 244 (44.9%) were positive for NV. One hundred fifty-nine outbreaks (approximately 1,300 samples) were selected randomly from the individual years in proportion to the number of outbreaks that were positive in that year. The overall number of infected persons was 3,499, with a range of 5 to 150. In total, 316 samples from the 159 outbreaks were selected and sequenced (usually two samples per outbreak). The sequenced samples consisted of 13 outbreaks (26 samples) from 2001, 99 outbreaks (196 samples) from 2002, 35 outbreaks (70 sequences) from 2003, and 12 outbreaks (24 sequences) from 2004. NV testing was done by reverse transcriptase PCR (RT-PCR).
RNA extraction. Viral RNA was extracted from 300 μl of 20% stool suspensions by using size-fractionated silica particles (Sigma-Aldrich Steinheim, Germany) to bind the RNA in the presence of guanidinium isothiocynate (6). After being washed in a guanidinium-containing buffer, the RNA was eluted in nuclease-free water containing 40 U RNase inhibitor (RNasin; Promega, Madison, Wis.) and was either reverse transcribed immediately or stored at –70°C.
RT-PCR. A nested RT-PCR method was used for testing RNA from stool samples for NV, using the primer pair NV32 (5'-4226-4245ATG AAT ATG AAT GAA GAT GG-3') and NV36 (5'-4707-4726ATT GGT CCT TCT GTT TTG TC-3') for first-round amplification and primer pair NV33 (5'-4280-4299TAC CAC TAT GAT GCA GAT TA-3') and NV35 (5'-4617-4636GTT GAC ACA ATC TCA TCA TC-3') for second-round amplification (numbers refer to the nucleotide positions of Lordsdale virus [GenBank accession no. X86557] [33]). The final product is a 338-bp portion of the RdRp gene of both genogroups I and II. Briefly, 5 μl of the extracted RNA was added to a 15-μl mix containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, each deoxynucleoside triphosphate (Amersham Pharmacia Biotech, Piscataway, NJ) at a concentration of 1 mM, and 20 U of avian myeloblastoma virus reverse transcriptase (Roche Applied Science, Mannheim, Germany). Reverse transcription was performed at 25°C for 10 min, followed by 42°C for 60 min, at the end of which the reaction was stopped by denaturing the enzyme at 98°C for 5 min and the mixture was cooled down to 4°C. Two microliters of the resulting cDNA was added to 48 μl of the PCR mix in a 50-μl reaction mixture consisting of 10 mM Tris-HCl, 50 mM KCl, 1.25 mM MgCl2, 2.5 U of Taq polymerase (Promega, Madison, Wis.), each deoxynucleoside triphosphate at a concentration of 125 μM, and 30 pmol each of the NV32 and NV36 primers. Amplification was performed with the following cycling profile: initial denaturation at 94°C for 60 s; 35 cycles of denaturation at 94°C for 30 s, annealing at 42°C for 30 s, and extension at 72°C for 45 s; and a final extension at 72°C for 3 min. The nested PCR was performed using 1 μl of the first PCR product under the same reaction conditions with primers NV33 and NV35. The final product was examined by gel electrophoresis in 2% agarose gels containing ethidium bromide.
Nucleotide sequencing. The 338-bp product from the RT-PCR was purified with the Qiaquick kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. For DNA sequencing reaction, the fluorescence-labeled dideoxynucleotide technology from Applied Biosystems was used. The sequenced fragments were separated, and the data were collected with an automated sequencer (model 310; Applied Biosystems, Foster City, CA).
Nucleotide sequence analysis. Sequences were aligned with the ClustalW program (34). Phylogenetic analyses were done with the PHYLIP 3.6 package (12), using the Jukes-Cantor distance and neighbor-joining methods. Statistical confidence for the evolutionary trees was assessed by bootstrap analysis (1,000 replicates) and trees drawn with the Treeview program (31). The sample numbers are presented in the phylogenetic tree, preceded by the year of the outbreak and the outbreak number. The Manchester strain of sapoviruses (25) was used as the outgroup; also included are 14 reference strains taken from GenBank and 8 previously published and unpublished strains, also from GenBank (7, 17, 19). The Rostock039 virus (accession no. AF312520), identified in Germany in 2000, was used to represent the Grimsby-like sequences.
Nucleotide sequence accession numbers. The nucleotide sequence data have been submitted to GenBank and assigned accession numbers DQ157056 to DQ157140.
RESULTS
Outbreaks occurred in all parts of Baden-Württemberg and in many different settings, which can be grouped as shown in Table 1. Most of the outbreaks took place in medical facilities (66.7%; n = 106 outbreaks), followed by restaurants and catering facilities (17%; n = 27 outbreaks), facilities for children and teenagers (11.9%; n = 19 outbreaks), and private celebrations (3.1%; n = 5 outbreaks).
The distribution of the outbreaks within the study period shows that outbreaks occurred in all the months of 2002 but not in all months in other years (Fig. 1). Furthermore, there were also seasonal differences observed; 72% of all outbreaks occurred in autumn and winter, from October to March.
Ninety-nine (62.3%; n = 159) of the outbreaks took place in 2002. In this year, 35 outbreaks occurred in December alone. Most of the outbreaks in 2003 took place at the beginning of the year, with 13 outbreaks in the month of January alone. These could be seen as a part of the epidemic that started in the previous year.
To determine the genetic diversity of the noroviruses studied, 282-nucleotide stretches from the polymerase regions of 316 samples obtained from 159 outbreaks were sequenced. Pairwise alignment of two sequences from each outbreak showed that the sequences of noroviruses from 119 outbreaks (74.8%) were exactly the same, noroviruses from 13 outbreaks (8.1%) had one or two nucleotide differences, those from six outbreaks (3.8%) had more than two nucleotide differences despite repetition of sequencing, those from nine outbreaks (5.7%) could not be compared (only one sample from each of these outbreaks was successfully sequenced), and those from eight outbreaks (5%) had nonreproducible results (that is, one or both sequences were different on repeated sequencing). Although this phenomenon has already been reported in other studies (21), the results were regarded as inconclusive. Furthermore, there were four outbreaks from which only one sample per outbreak was obtained. Out of the 316 samples sequenced, 265 sequences, including 25 sequences from 2001, 153 sequences from 2002, 63 sequences from 2003, and 24 sequences from 2004, were used to generate a phylogenetic tree (Fig. 2). In all, sequences from 148 outbreaks were used for the alignment.
The sequence analysis shows that genogroup II was predominant in all of the years, accounting for 95.3% of the outbreaks used for the phylogenetic tree and 9 of the 12 clusters in which a sequence(s) from this study was included. There were seven clusters in 2001, eight clusters in 2002, six in 2003, and four in 2004 when the phylogenetic trees for the individual years were reconstructed (results not shown), indicating a reduction in the number of circulating strains in 2003 and 2004. All of the sequences from the same outbreaks in 2001 and 2004 clustered together. However, the sequences from one outbreak (2.9%) in 2003 and two outbreaks (2.1%) in 2002 separated into different clusters, corresponding to the results of the pairwise alignment. This is despite the fact that only sequences of outbreaks in which there were fewer than three base differences after repeated sequencing were included in the phylogenetic analysis.
Forty-four sequences from 25 outbreaks clustered with the Suria and Castell viruses, which were identified by Buesa et al. (7) and designated GGIIb. A predominant number of the outbreaks in 2001 (four outbreaks) clustered with Melksham virus and the Castell/Suria viruses. For the rest of the years, most of the outbreaks clustered with Camberwell virus (GGII/4). This was most obvious for 2002 and 2003, in which 102 (66.7%) and 47 (74.6%) of all the sequences in the phylogenetic tree belonged to this cluster, respectively. Out of the Camberwell cluster, 84 (82.4%) strains in 2002 and 43 (91.5%) strains in 2003 were found to have the unusual mutation of AACTTG to AATCTG at position 4820 of Norwalk virus (accession no. M87661) when related to the Grimsby and Grimsby-like viruses (26). "New variant" was used for the viruses identified with this mutation to differentiate them from the closely related Grimsby and Grimsby-like viruses. In all, 133 sequences forming 77.7% of the Camberwell cluster and 50.1% of all the sequences analyzed belonged to the variant. These groups of viruses were found at a lower percentage (25%) in 2004 and not found among all the sequences analyzed in 2001.
The 2001 and 2002 GGI strains did not completely resolve into any of the reference strains. The 2001 strain had a bootstrap value of 270 with Southampton virus (accession no. L07418), its closest reference strain, while the 2002 strain had a bootstrap value of 419 with BS5 virus (accession no. AF093797), its closest reference strain. However, the 2003 and 2004 strains clustered with the Southampton (bootstrap value, 1,000) and Norwalk (bootstrap value, 994) viruses, respectively (results not shown). In the general tree (Fig. 2), the unresolved 2001 and 2002 strains clustered together, with a bootstrap value of 365 with BS5 virus, their common closest reference strain, while for 2003 and 2004, all strains clustered the same as in the individual trees. The GGI clusters had a similarity of 92 to 100% for nucleotide sequences within individual groups, while the GGII clusters had a similarity of 78 to 100% for nucleotide sequences within the groups for the analyzed region. Among all the clusters, the nucleotide sequence similarities were 74 to 100% for GGI, 60 to 100% for GGII, and 49 to 67% between the genogroups (Table 2).
Some virus strains were found to have been involved in more than one outbreak in the same area, and circulation of more than one strain at the same place in the same year was also seen. There was also cocirculation of more than one genotype at the same period, as outbreaks that were reported in the same month and period were found in different clusters.
DISCUSSION
Samples from outbreaks of acute gastroenteritis from the state of Baden-Württemberg are sent to the State Health Office in Stuttgart on a regular basis. Our results show that NV account for 44.9% of all outbreaks of gastroenteritis reported during the study period. A representative number of these outbreaks were selected for further study of their epidemiological characteristics.
Our results showed that medical facilities accounted for 66.7% of the outbreaks and 70.2% of the persons infected in this study. This was followed by outbreaks in food outlets (restaurants and catering facilities), which accounted for 17% of the outbreaks. In an earlier study in Germany, all settings making up medical facilities together accounted for over 90% of all outbreaks studied (32), while in the United Kingdom, hospitals as the only medical facility accounted for 86.7% of all outbreaks studied (27). Both values are higher than the 66.7% we recorded; however, our value was higher than the 43% share of outbreaks in medical facilities reported in the United States (11).
As seen from the phylogenetic tree, the GGII strains of the virus were found to be responsible for most (95.3%) of the outbreaks. Studies from different parts of the world have also reported the predominance of GGII, e.g., in France (5), The Netherlands (24), the United Kingdom (27), Australia (23), Canada (16), and the United States (11, 29), with many of them in the same range as our value. Among the GGI strains identified in this study, two outbreaks (outbreak 151 in 2001 and outbreak 007 in 2002) clustered together but failed to completely resolve into any of the reference strains used. This may be attributed to the circulation of a new strain or to the position of the genome used in the study. While the RNA polymerase region is mostly used for diagnosis of NV (3), a complete identification requires the use of the capsid region (21).
The identification of many different virus strains, belonging to 12 clusters within the 40 months of this study, shows that many genotypes were circulating in the region during this period. An increase in the number of clusters was observed in 2002 compared to 2001. Thereafter, there was a gradual decrease in the number of clusters circulating over the years. The minimum percent similarity within the Camberwell virus group (78%), which is below the 90% expected for GGII within the polymerase region (36), indicates that the group may contain more than one genotype.
In this study we found the involvement of more than one strain of virus in the same outbreak, indicating that two different genotypes may have been involved. This was further confirmed by sequencing more amplicons from three selected outbreaks with this pattern (results not shown). There were also cases where two different sequences were obtained from the same stool sample, but the results were inconclusive and the sequences were not included in the analysis. The existence of multiple sequences in an outbreak is usually associated with shellfish-related outbreaks due to the ability of shellfish to concentrate NV from a contaminated environment as a result of their filter-feeding mechanism (20). However, some other studies have reported the presence of multiple sequences in outbreaks not related to shellfish and even in sporadic cases (7, 21). The circulation of more than one strain in the same place raises the possibility of more than one virus strain being involved in the same outbreak.
The GGII4 "new variant" (26), which clustered with the Camberwell virus (Fig. 2), first emerged in 2002 and became the predominant circulating strain in the same year. The same variant of virus was also identified as the predominant strain in other European countries (26). Incidentally, Rostock039, isolated in Germany in 2000 (accession no. AF312520) (unpublished report), had the Grimsby-like earlier sequence of AACTTG. Although this group of viruses is said to have been responsible for the epidemic outbreak of acute gastroenteritis that was observed in 2002 and spilled over into the early part of 2003, it does not appear as if it will remain endemic as reported for Grimsby virus in the United Kingdom (15). Not only did the number of outbreaks attributed to this virus start going down in 2003, but the percentage of outbreaks caused by the group fell dramatically from 68.3% in 2003 to 25% in 2004.
The Castell and Suria strain group, which has been reported in various parts of Europe and was also reported in drinking water in Sweden (30), was also found in all the years of this study. This group and the Melksham virus group were the predominant strains in 2001. The Castell/Suria strains were reported to be related to Mexico virus by capsid analysis (7). Mexico virus was part of the group identified to have been replaced at the beginning of 2002 by the new GGII4 variant in Europe (26).
In this study, we have been able to demonstrate clearly that NV are an important cause of gastroenteritis in Germany. We showed that there are more than three genotypes involved in causing outbreaks of acute gastroenteritis throughout the period of this study. More work needs to be done to characterize the virus strains identified in this study fully and to understand the involvement of multiple sequences in individual infections and outbreaks. The results of this work will contribute to a better understanding of the epidemiology and pathogenicity of NV in Germany.
ACKNOWLEDGMENTS
We are grateful to all the staff of the molecular laboratory of the State Health Office Stuttgart and particularly to Christine Herrmann and Elke Gutwein for some of the laboratory work.
This study was partially supported by the Landesstiftung Baden-Württenberg gGmbH, Stuttgart, Germany (grant P-LS-E/MOP A8-8).
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