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Journal of Clinical Microbiology, October 2008, p. 3397-3403, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.01932-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Single Base Substitutions in the Capsid Region of the Norovirus Genome during Viral Shedding in Cases of Infection in Areas Where Norovirus Infection Is Endemic

Mayumi Obara,¹ Sumiyo Hasegawa,¹ Masae Iwai,¹ Eiji Horimoto,¹ Kazuya Nakamura,¹ Takeshi Kurata,¹ Naohito Saito,² Hiroshi Oe,² and Takenori Takizawa^1*

Department of Virology, Toyama Institute of Health, Imizu, Toyama 939-0363,¹ Niikawa Health Center, Kurobe, Toyama 938-0025, Japan²

Received 1 October 2007/ Returned for modification 7 November 2007/ Accepted 24 July 2008

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Norovirus (NoV) infections are the major cause of food- and waterborne nonbacterial gastroenteritis in Japan. Some individuals showed long-term excretion of the virus into feces in 29 outbreaks of acute nonbacterial gastroenteritis that occurred in Toyama Prefecture, Japan, in fiscal year 2006. In one of these cases, single base substitutions from A to G in the capsid region of the NoV genome were commonly detected in two individuals during virus shedding by direct sequencing of PCR products. The A-to-G substitution was accompanied by an N-to-S amino acid change. The population of clones that possessed A at the corresponding site was gradually replaced by those with G during the infectious course. Although other substitutions were observed in the complete open reading frame 2 sequence, they were not common in these two individuals. NoVs are capable of evolving in the gastroenteric tract.

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Noroviruses (NoVs), previously known as Norwalk-like virus or small round structured virus, belong to the family Caliciviridae. The NoV genome is a single-stranded, positive-sense RNA molecule of about 7.5 kb that comprises three open reading frames (ORFs) (6). NoV infections are the major cause of acute nonbacterial gastroenteritis worldwide, and the illness occurs in people of all ages. NoVs are transmitted not only by the fecal-oral route but also by direct person-to-person contact. Numerous outbreaks due to NoV-contaminated food and water have been reported (8, 11, 15, 26).

Since no in vitro culture system for NoVs has been established, electron microscopy (EM) or immuno-EM is routinely used to diagnose infections and to detect NoV particles in stool specimens. After the cloning and sequencing of representative NoV strains, Norwalk/68/US (14) and Southampton/91/UK (20), a reverse transcription-PCR (RT-PCR) assay was developed to target the RNA-dependent RNA polymerase gene of ORF1 of the NoV genome (2, 3, 13). Based on sequence information obtained from the polymerase region, human NoV strains can be divided into three genogroups: genogroup I (GI), GII, and GIV (23). NoV GI and GII each comprise a large number of genetically diverse strains (4, 12). A recent study indicated that NoV GI and GII strains consist of at least 14 and 17 genotypes, respectively (17, 23).

ORF2 encodes a major structural capsid protein, including a shell (S) domain and a protruding (P) domain (1, 24). Several reports have suggested a correlation between the genetic clustering of ORF2 and antigenicity confirmed by the patient's immune response (7, 22). It has been suggested that considerable genetic and antigenic divergence is a major cause of the repetitive global prevalence of this virus (9, 27). Despite the significance of the variation, the origin of the diversity and evolutionary mechanisms by which new and possibly more virulent strains evolve remain unclear. Recent studies have shown that the virus can be excreted from a person for much longer than previously thought (25), and nucleotide as well as amino acid changes accumulate, especially in the P2 domain of the capsid region, suggesting immune-mediated positive selection (21). Here, we report an NoV-infected group, in which two employees shed viruses in feces for up to 2 months. During excretion of the virus, a certain amino acid change resulting from a single nucleotide substitution in the capsid region was observed, suggesting evolution of the virus in the gastrointestinal tract.

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Outbreaks and specimens. Between April 2006 and March 2007, 29 outbreaks of acute nonbacterial gastroenteritis occurred in Toyama Prefecture, Japan. NoV GII and NoV GI genotypes were detected in 28 cases and 1 case, respectively, by real-time PCR. The outbreak described in the present study occurred in a local hotel in May 2006. Clinical information and stool specimens were collected from 30 individuals, and the samples were examined for microorganisms.

RNA extraction and RT. A 10% stool suspension was prepared as described previously (16). Viral RNA was extracted from 140 µl of the suspension with a QIAamp Viral RNA minikit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA was eluted with 60 µl of elution buffer. Viral RNA (12 µl) was treated at 37°C for 30 min and then 75°C for 5 min in 3 µl of DNase mixture (1 U of DNase I, 125 mM Tris-HCl [pH 8.3], 187.5 mM KCl, and 7.5 mM MgCl₂). This was followed by the addition of 15 µl of a mixture containing 75 mM Tris-HCl (pH 8.3), 112.5 mM KCl, 4.5 mM MgCl₂, a 1 mM concentration of each deoxynucleoside triphosphate, 10 mM dithiothreitol, 0.75 µg of random hexamer [pd(N)₆; GE Healthcare, Piscataway, NJ], 33 U of RNase inhibitor (Takara Bio Inc., Otsu, Japan), and 300 U of SuperScript II RNase H^– transcriptase XL (Invitrogen Corp., Carlsbad, CA). RT was performed at 42°C for 60 min, and the enzyme was inactivated at 99°C for 5 min.

Real-time PCR. Real-time PCR was carried out as described by Kageyama et al. (16). Real-time quantitative PCR was carried out in 50-µl reaction mixtures containing 5 µl of cDNA; 25 µl of TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ); 20 pmol each of the primers COG2F (5'-CAR GAR BCN ATG TTY AGR TGG ATG AG), ALPF (5'-TTT GAG TCC ATG TAC AAG TGG ATG CG), and COG2R (5'-TCG ACG CCA TCT TCA TTC ACA); and 11.4 pmol of probe RING2AL-TP (5'-VIC-TGG GAG GGS GAT CGC RAT CT-TAMRA) for the detection of GII NoV. PCR amplification was performed with an ABI 7000 sequence detector (Applied Biosystems) under the following conditions: 2 min at 50°C and 10 min at 95°C and then 45 cycles at 95°C for 15 s and 56°C for 1 min. The data were corrected by using internal standards as described by Kageyama et al. (16).

PCR. To amplify the N-terminal/shell (N/S) domain of the capsid region, PCR was carried out with puRe Taq Ready-To-Go PCR beads (GE Healthcare) and the primers G2-SKF (5'-CNT GGG AGG GCG ATC GCA A) and G2-SKR (5'-CCR CCN GCA TRH CCR TTR TAC AT) for GII NoV strains as described by Kojima et al. (19). The mixture containing 2 µl of cDNA was incubated at 95°C for 10 min, followed by 40 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min, with a final incubation of 72°C for 15 min.

To amplify ORF2, PCR was carried out with 50 pmol primers LV6717 (5'-AGT ACC TTG TTC CGC TCC A) and LV4922G10A (5'-CAC GGC CCA ACA TTC TAC) modified from the reference (21). A 50-µl PCR mixture contained 2 µl of cDNA, 2.5 U of TaKaRa LA Taq (Takara Bio), 10x LA PCR buffer II 5 µl, 2.5 mM MgCl₂, and a 0.4 mM concentration of each deoxynucleoside triphosphate. Amplification was performed by using 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min. Second PCR was performed in the same way.

Sequencing analysis. After purification of the amplicon, the nucleotide sequences were determined with the BigDye Terminator v3.1 cycle sequencing kit and an ABI 3100 or 3130 sequencer (Applied Biosystems).

Cloning analysis. PCR products of the N/S domain were cloned by using a TOPO10 PCR cloning kit (Invitrogen) according to the manufacturer's instructions. Plasmid DNA was purified by using a mini-plasmid purification kit (Promega Corp., Madison, WI) according to the manufacturer's recommendations. Plasmids containing an insert of appropriate length were sequenced as described above with the M13 universal primer.

Phylogenetic analysis. Capsid sequences of the reference strains of NoV were obtained from GenBank. Phylogenetic analysis was performed as described by Katayama et al. (17). Briefly, 302-bp sequences of the N/S domain of the capsid region were aligned by using CLUSTAL W (version 1.83). A phylogenetic tree was constructed by the neighbor-joining method, and genetic distances were calculated according to the Kimura two-parameter method (18). The reliability of the tree was estimated by performing 1,000 bootstrap replications, and bootstrap values of 950 or higher were considered statistically significant for a grouping (10).

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Outbreak. An outbreak of gastroenteritis occurred in a local hotel in Toyama prefecture. Fecal specimens from thirty individuals, including patients and employees, were collected and examined for microorganisms. Real-time PCR analysis identified the GII sequence in specimens from fourteen individuals. Consecutive surveys showed that two employees excreted the NoV gene for one and two months, respectively, even after their symptoms had disappeared (Table 1 and Fig. 1). The copy number of the genome in one employee showed two characteristic peaks, followed by a rapid decrease to below the limit of detection ("a" terms in Fig. 1). The other asymptomatic employee showed a continuous decrease in the copy number of the NoV genome to an undetectable level ("b" terms in Fig. 1).

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TABLE 1. Detection of NoVs from employees

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FIG. 1. Change in copy numbers of NoVs in feces obtained from employees. The copy number of NoV was measured by real-time PCR, and represented by the log10 copy number/g of feces. Open symbols indicates NoV negativity. The horizontal bold line indicates the limit of detection.

Sequence analysis. Sequencing revealed that the NoV causing this outbreak belonged to GII genotype 4, which is the most predominant type in recent outbreaks in Japan (5). Phylogenetic analysis showed that recently identified sequences, including this one, formed a distinct cluster from past-identified sequences of genotype 4 (Fig. 2). Direct sequence analyses of consecutive specimens from these two employees revealed that two single base substitutions occurred: (i) A (nucleotide 50 counting from the first initiation codon of the capsid gene) changed to G and (ii) C (nucleotide 279) to T. The C-to-T change was detected only in subject "a" between the first and second consecutive specimens, while the A-to-G change was observed in both subjects a and b, between the third and fourth and between the first and second specimens, respectively. Although no signal from "a-1" was detected by real-time PCR (Table 1), the PCR product was not regarded as a false-positive reaction due to contamination, since patient a was already symptomatic on May 10. The sequences of specimens from another 11 of 12 individuals in this outbreak had A (nucleotide 50) and T (nucleotide 279). The remaining individual could not be analyzed.

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FIG. 2. Phylogenetic tree of NoV GII genotype 4 sequences obtained from outbreaks in Toyama prefecture from April 2003 to March 2007. Symbols indicate the following seasons: , 2003/2004; •, 2004/2005; , 2005/2006; , 2006/2007. The shaded area indicates outbreaks that occurred in the 2006/2007 season. Samples except those from cases 3 and 4 in the 2006/2007 season form a unique cluster. Samples from cases 3 and 4 form another unique cluster. Samples are showed as "case no._month/year" (mass outbreaks) or as "sample no._month/year." The case that involves employees a and b is indicated by an arrow. Reference strains are underlined and presented as "strain (accession no.)".

To examine whether additional nucleotide change existed in another area of ORF2, especially in the hypervariable region, we determined the complete sequence of ORF2 in these specimens. The ORF2s in the specimens from a-2, a-6, b-1, and b-2 were amplified by PCR and directly sequenced. Alignment of the sequences of ORF2 (1,623 bp) revealed that eight single base substitutions occurred (including the A-to-G change at nucleotide 50) and one microheterogeneity occurred in a-2 at 209, which was recognized by the superimposed signals in the sequencing reaction (Table 2). Only the substitution at nucleotide 50 was common in the two employees. A different nucleotide between a-2 and b-1 already existed at nucleotide 516. All of these nucleotide changes result in amino acid changes. One amino acid change at position 17 is located in the N-terminal domain, four changes at positions 70, 108, 172 and 193 are located in the S domain, two changes at positions 291 and 297 are located in the P2 domain, and another two at positions 460 and 483 are located in the P1 domain (Fig. 3); thus, the amino acid changes were not restricted to some distinct region.

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TABLE 2. Alignment of the sequence of the complete ORF2 region of NoV GII from employees a and b

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FIG. 3. Alignment of the amino acid sequence of ORF2. Amino acids identical to the consensus sequence are indicated by periods, and only the different sequences are included. The positions of S, P1, and P2 domains are estimated (1, 24) and shown above amino acid number. The codon at amino acid 70 of a-2 is N (AAC) or S (AGC) and is indicated by "?".

Cloning analysis. To assess whether clonal change of the viral genome occurred in these cases, the PCR products were each cloned into plasmid DNA, and 20 to 30 clones were isolated and sequenced. As for the A-to-G change, while all a-2 and a-3 clones had A at this position, 9 and 15 clones of a-4 had A and G, respectively. One and twenty-four clones of a-5 had A and G, respectively, and all clones of a-6 had G (Fig. 4A and Table 3). In the case of subject b, all clones of b-1 had A, whereas 1 and 28 clones of b-2 had A and G, respectively (Table 3). Thus, nucleotide 50 in the capsid region seemed to gradually change from A to G during the infectious course of both individuals.

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FIG. 4. Nucleotide sequences of the cloned capsid region. (A) Alignment of cloned nucleotide sequences from positions 41 to 60 of consecutive specimens of employee a. PCR products were cloned into plasmid DNA and sequenced. The consensus sequence is shown at the top of the figure. Only nucleotides different from the consensus sequence are included. (B) Alignment of cloned sequences from positions 61 to 80 of consecutive specimens of employee a as a representative of nucleotide variations.

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TABLE 3. Numbers of clones containing an A or G residue at nucleotide 50 and C or T residue at nucleotide 279 of employees a and b

As for the C-to-T change, all clones of a-1 possessed a C residue, whereas those of a-2 possessed T (Table 3); thus, no gradual change was observed.

Although other nucleotide variations or deletions among the clones were observed in the capsid region, none was fixed to a unique sequence (Fig. 4B). The A-to-G substitution changes amino acid number 17 of the capsid from N to S; however, the N-to-S substitution may not result in a significant structural alteration of the capsid protein, because the amino acid sequence surrounding 17N (N-L-V) does not match the consensus sequence of N-linked glycosylation (N-X-S/T), and capsid protein has not been reported to be glycosylated. The C-to-T change does not cause an amino acid change.

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In this report, we examined the sequence of the capsid region of NoV and found nucleotide and amino acid substitutions in consecutive specimens of two individuals that shed the virus into feces for a relatively long period of time.

Since an A-to-G change occurring in two individuals at the same time is unlikely, the change likely occurred in one of them, and then the mutated NoV was transmitted to the other. The proportion of b-2 (on June 5) clones with G (A:G = 1:28) was higher than that of a-4 (A:G = 9:15) (on June 6). The second increase in the copy number of the NoV genome in employee a on June 20 also indicates that the NoV in employee b with very high copy numbers was transmitted to employee a at this time.

NoV with a T residue might have been transmitted from subject b to subject a on around 10 May to cause the first increase in the copy number, and took over the NoV with C in subject a within a short period. Since all other NoV genomes detected in Toyama prefecture in 2005 and 2006 had a C residue at this site, the NoV with T is unique in recent outbreaks. That subject b demonstrated extremely high copy numbers of NoV, especially in the early phase, indicates that multiple replications of NoV occurred in this individual; therefore, the C-to-T change might have occurred in subject b and been transmitted to subject a.

Since the outbreak occurred in an isolated area in Toyama prefecture, the possibility that different NoV was introduced from another area seems to be extremely low. Although the NoVs in subjects a and b were possibly transmitted to each other, only one with a high copy number seems to have affected the other with a low copy number.

Other different nucleotides found in a-6 and b-2 may be the consequence of independent evolution in these individuals. The higher number of changes in a-6 than those in b-2 may reflect a longer incubation time in the gastroenteric tract. Other sporadic variations observed in both the capsid and the polymerase region (data not shown) did not persist during the infectious course. Although the possibility remains that a small population of NoV with G at the corresponding position already existed at the first exposure, our observations suggest that NoV really evolves in the gastrointestinal tract in a single individual.

An accumulation of mutations in the capsid region was reported in a persistently NoV-infected patient who excreted the virus for more than 2 years (21). Most of the mutations were accumulated in the hypervariable domain (P2 domain), which is the most exposed part of the structure and has been proposed to contain determinants for strain specificity (24). Hence, these mutations were speculated to be a result of immune pressure. The N-to-S change in the capsid protein caused by the A-to-G nucleotide change in this report is located at amino acid 17, as counted from the N terminus of the protein, where an apparent antigenic epitope has not been observed (28). Consistently, other changes found in ORF2 were not restricted to the hypervariable P2 domain. Thus, these changes seem not to be caused by immune-driven pressure. Mechanisms other than immune pressure, such as interaction with receptors, or a relation to other changes elsewhere in the genome of NoV, might be associated with amino acid substitution. Alternatively, these changes might be only footprints that reflect the viral evolution occurring in the gastroenteric tract.

 
We thank Miyuki Maekawa for technical assistance.

This study was supported by a Health Labor Sciences Research Grant in Research on Emerging and Re-emerging Infectious Disease from the Japanese Ministry of Health, Labor, and Welfare.

 
* Corresponding author. Mailing address: Department of Virology, Toyama Institute of Health, 17-1 Nakataikoyama, Imizu-shi, Toyama 939-0363, Japan. Phone: 81-766-56-8143. Fax: 81-766-56-7326. E-mail: takenori.takizawa{at}pref.toyama.lg.jp

Published ahead of print on 6 August 2008.

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1
1. Allen, D. J., J. J. Gray, C. I. Gallimore, J. Xerry, and M. Iturriza-Gómara. 2008. Analysis of amino acid variation in the P2 domain of the GII-4 norovirus VP1 protein reveals putative variant-specific epitopes. PLoS ONE 3:e1485.[CrossRef]
2
2. Ando, T., S. S. Monroe, J. R. Gentsch, Q. Jin, D. C. Lewis, and R. I. Glass. 1995. Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization. J. Clin. Microbiol. 33:64-71.[Abstract]
3
3. Ando, T., Q. Jin, J. R. Gentsch, S. S. Monroe, J. S. Noel, S. F. Dowell, H. G. Cicirello, M. A. Kohn, and R. I. Glass. 1995. Epidemiologic applications of novel molecular methods to detect and differentiate small round structured viruses (Norwalk-like viruses). J. Med. Virol. 47:145-152.[Medline]
4
4. Ando, T., J. S. Noel, and R. L. Fankhauser. 2000. Genetic classification of "Norwalk-like viruses." J. Infect. Dis. 181(Suppl. 2):S336-348.[CrossRef][Medline]
5
5. Anonymous. 2007. Infectious Disease Surveillance Center prompt report. Infectious Disease Surveillance Center, Tokyo, Japan. http://idsc.nih.go.jp/iasr/noro.html.
6
6. Atmar, R. L., and M. K. Estes. 2001. Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin. Microbiol. Rev. 14:15-37.[Abstract/Free Full Text]
7
7. Belliot, G., J. S. Noel, J.-F. Li, Y. Seto, C. D. Humphrey, T. Ando, R. I. Glass, and S. S. Monroe. 2001. Characterization of capsid genes, expressed in the baculovirus system, of three new genetically distinct strains of "Norwalk-like viruses." J. Clin. Microbiol. 39:4288-4295.[Abstract/Free Full Text]
8
8. Daniels, N. A., D. A. Bergmire-Sweat, K. J. Schwab, K. A. Hendricks, S. Reddy, S. M. Rowe, R. L. Fankhauser, S. S. Monroe, R. L. Atmar, R. I. Glass, and P. Mead. 2000. A foodborne outbreak of gastroenteritis associated with Norwalk-like viruses: first molecular traceback to deli sandwiches contaminated during preparation. J. Infect. Dis. 181:1467-1470.[CrossRef][Medline]
9
9. Dingle, K. E., et al. 2004. Mutation in a Lordsdale norovirus epidemic strain as a potential indicator of transmission routes. J. Clin. Microbiol. 42:3950-3957.[Abstract/Free Full Text]
10
10. Efron, B., E. Halloran, and S. Holmes. 1996. Bootstrap confidence levels for phylogenetic trees. Proc. Natl. Acad. Sci. USA 93:13429-13434.[Abstract/Free Full Text]
11
11. García, C., H. L. DuPont, K. Z. Long, J. I. Santos, and G. Ko. 2006. Asymptomatic norovirus infection in Mexican children. J. Clin. Microbiol. 44:2997-3000.[Abstract/Free Full Text]
12
12. Green, J., J. P. Norcott, D. Lewis, C. Arnold, and D. W. G. Brown. 1993. Norwalk-like viruses: demonstration of genomic diversity by polymerase chain reaction. J. Clin. Microbiol. 31:3007-3012.[Abstract/Free Full Text]
13
13. Green, J., C. I. Gallimore, J. P. Norcott, D. Lewis, and D. W. Brown. 1995. Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV-associated gastroenteritis. J. Med. Virol. 47:392-398.[Medline]
14
14. Jiang, X., D. Y. Graham, K. Wang, and M. K. Estes. 1990. Norwalk virus genome cloning and characterization. Science 250:1580-1583.[Abstract/Free Full Text]
15
15. Johansson, P. J. H., M. Torvén, A.-C. Hammarlund, U. Björne, K.-O. Hedlund, and L. Svensson. 2002. Food-borne outbreak of gastroenteritis associated with genogroup I calicivirus. J. Clin. Microbiol. 40:794-798.[Abstract/Free Full Text]
16
16. Kageyama, T., S. Kojima, M. Shinohara, K. Uchida, S. Fukushi, F. B. Hoshino, N. Takeda, and K. Katayama. 2003. Broadly reactive and highly sensitive assay for Norwalk-like viruses based on real-time quantitative reverse transcription-PCR. J. Clin. Microbiol. 41:1548-1557.[Abstract/Free Full Text]
17
17. Katayama, K., H. Shirato-Horikoshi, S. Kojima, T. Kageyama, T. Oka, F. B. Hoshino, S. Fukushi, M. Shinohara, K. Uchida, Y. Suzuki, T. Gojobori, and N. Takeda. 2002. Phylogenetic analysis of the complete genome of 18 Norwalk-like viruses. Virology 299:225-239.[CrossRef][Medline]
18
18. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.[CrossRef][Medline]
19
19. Kojima, S., T. Kageyama, S. Fukushi, F. B. Hoshino, M. Shinohara, K. Uchida, K. Natori, N. Takeda, and K. Katayama. 2002. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J. Virol. Methods 100:107-114.[CrossRef][Medline]
20
20. Lambden, P. R., E. O. Caul, C. R. Ashley, and I. N. Clarke. 1993. Sequence and genome organization of a human small round-structured (Norwalk-like) virus. Science 259:516-519.[Abstract/Free Full Text]
21
21. Nilsson, M., K.-O. Hedlund, M. Thorhagen, G. Larson, K. Johansen, A. Ekspong, and L. Svensson. 2003. Evolution of human calicivirus RNA in vivo: accumulation of mutations in the protruding P2 domain of the capsid leads to structural changes and possibly a new phenotype. J. Virol. 77:13117-13124.[Abstract/Free Full Text]
22
22. Noel, J. S., T. Ando, J. P. Leite, K. Y. Green, K. E. Dingle, M. K. Estes, Y. Seto, S. S. Monroe, and R. I. Glass. 1997. Correlation of patient immune responses with genetically characterized small round-structured viruses involved in outbreaks of nonbacterial acute gastroenteritis in the United States, 1990 to 1995. J. Med. Virol. 53:372-383.[CrossRef][Medline]
23
23. Phan, T. G., K. Kaneshi, Y. Ueda, S. Nakaya, S. Nishimura, A. Yamamoto, K. Sugita, S. Takanashi, S. Okitsu, and H. Ushijima. 2007. Genetic heterogeneity, evolution, and recombination in noroviruses. J. Med. Virol. 79:1388-1400.[CrossRef][Medline]
24
24. Prasad, B. V. V., M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes. 1999. X-ray crystallographic structure of the Norwalk virus capsid. Science 286:287-290.[Abstract/Free Full Text]
25
25. Rockx, B., M. De Wit, H. Vennema, J. Vinjé, E. De Bruin, Y. Van Duynhoven, and M. Koopmans. 2002. Natural history of human calicivirus infection: a prospective cohort study. Clin. Infect. Dis. 35:246-253.[CrossRef][Medline]
26
26. Sasaki, Y., A. Kai, Y. Hayashi, T. Shinkai, Y. Noguchi, M. Hasegawa, K. Sadamasu, K. Mori, Y. Tabei, M. Nagashima, S. Morozumi, and T. Yamamoto. 2006. Multiple viral infections and genomic divergence among noroviruses during an outbreak of acute gastroenteritis. J. Clin. Microbiol. 44:790-797.[Abstract/Free Full Text]
27
27. Vinjé, J., J. Green, D. C. Lewis, C. I. Gallimore, D. W. Brown, and M. P. Koopmans. 2000. Genetic polymorphism across regions of the three open reading frames of "Norwalk-like viruses." Arch. Virol. 145:223-241.[CrossRef][Medline]
28
28. Yoda, T., Y. Terano, Y. Suzuki, K. Yamazaki, I. Oishi, E. Utagawa, A. Shimada, S. Matsuura, M. Nakajima, and T. Shibata. 2000. Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli. Microbiol. Immunol. 44:905-914.[Medline]

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Journal of Clinical Microbiology, October 2008, p. 3397-3403, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.01932-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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