Molecular Cloning Expression Self-Assembly, Antigenicity, and Seroepidemiology of a Genogroup II Norovirus Isolated in France

Microbiologie Médicale et Moléculaire, Facultés de Médecine et Pharmacie, Université de Bourgogne, 21033 Dijon cedex, France

Received 18 March 2002/ Returned for modification 20 July 2002/ Accepted 24 April 2003

	ABSTRACT

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Virus-like particles of Dijon171/96 virus, a genogroup II norovirus, were expressed in a baculovirus system and were used for a seroepidemiological study of 1,078 age-stratified human sera collected in Dijon, France. The results showed a seroprevalence of 74.1%. Furthermore, we showed that murine antibodies generated against recombinant Dijon171/96 virus, and human antibodies recognized discontinuous epitopes on the particles.

	TEXT

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Norwalk-like viruses recently designated noroviruses (Caliciviridae family) represent the most important cause of acute gastroenteritis outbreaks in industrialized countries (8, 17) and are also now recognized as a frequent agent of gastroenteritis in the community in all age groups (4, 5, 19). Noroviruses are divided into two genogroups, genogroup I and genogroup II, each including different genotypes or genetic clusters (1, 20). Expression of Norwalk virus (NV) ORF2, encoding the major capsid protein, in insect cells infected with recombinant baculovirus has been reported previously (15). The capsid protein obtained self-assembled spontaneously into virus-like particles (VLPs) which are morphologically and antigenically similar to the native particles. VLPs have been subsequently prepared for different strains, notably Southampton genotype I (18), Hawaii genotype II (10), Mexico genotype II (14), Toronto GII (16), and Lordsdale genotype II (6). Because noroviruses are difficult to propagate, these VLPs represent an important source of antigen that can be used in place of the native virus to study seroprevalence and to better understand immunity to caliciviruses. The purpose of this study was to clone and express the recombinant capsid protein of a genogroup II Grimsby-like strain (Dijon171/96) (Lordsdale genotype) detected in France in a child during the winter of 1995-1996. The VLPs obtained were used in a seroepidemiological study in the population conducted between February 2000 and June 2001. In addition, we showed that mouse antibodies generated against recombinant Dijon171/96 (rDijon171/96) as well as human antibodies recognized discontinuous epitopes on the VLPs.

RNA was extracted from a stool specimen (Dijon171/96) with QiaAmp viral RNA kit (Qiagen). cDNA was obtained by using primer 2721 (nucleotides 7232 to 7255 in ORF3 [3]) and Superscript II RNase H (Life Technologies) according to the manufacturer's conditions. A nested PCR was used to amplify the entire ORF2 gene. The first PCR was performed with primer NI in ORF1 (positions 4768 to 4788 [9]), primer 2721, and Taq Pwo polymerase (Roche Molecular Biochemicals). The amplified product was sequenced, and a second PCR allowing the generation of the amplified ORF2 was carried out with the following primers, including EcoRI and BglII restriction sites (underlined): forward primer, 5'⁵⁰⁶⁸-GGCTCCCAGAATTCTGAATG-3'⁵⁰⁸⁹, and reverse primer 5'-⁶⁷⁰²CAAAGAGATCTCCAGCCATTA-3'⁶⁷²². A 1,620-bp fragment was cloned into the baculovirus transfer vector pVL1393 (Invitrogen). The ORF2 sequence predicted a 539-amino-acid capsid, which exhibited 98.7% nucleotide and 98.7% amino acid identity with Grimsby virus and exhibited 91.9% nucleotide and 96.1% amino acid identity with Lordsdale virus. Sf9 (Spodoptera frugiperda) insect cells were cotransfected with baculovirus linear DNA (Autographa californica nuclear polyhedrosis virus) and a recombinant plasmid (baculoGold kit; Pharmingen). At 5 days posttransfection the cells were harvested and a single recombinant virus clone was selected from the supernatant by plaque purification.

Production of the capsid protein was performed by infecting Sf9 cells at a multiplicity of infection of 1 and harvesting of cell cultures at 5 days postinfection. Purification was performed separately from both supernatant and infected cells after two freeze-thaw cycles. The recombinant capsid was first concentrated by ultracentrifugation. The resulting pellets were centrifuged through a 40% (wt/wt) sucrose cushion for 2 h at 28,000 rpm (Beckman SW28 rotor) and then through a preformed CsCl gradient (28 to 36% [wt/wt] in water; 1.2644 to 1.3661 g/cm³) for 22 h at 35,000 rpm (LKB RPS56T rotor). The capsid protein was identified in CsCl fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy examination after negative staining with 2% phosphotungstic acid, pH 7.0. VLPs were quantified by the Lowry method by using bovine serum albumin as a standard.

Peptides obtained from trypsinolysis of the 35-kDa protein purified from cell lysates were analyzed by Proteomic Solutions (France) with a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis after two-dimensional electrophoresis (13). Briefly, the purified CsCl fraction containing the 35-kDa protein (450 µg of protein) was mixed with sample buffer containing 9 M urea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 0.6% ampholytes, and 20 mM dithiothreitol. A pH linear gradient (3 to 10) was used for the first-dimension electrophoresis, and an SDS-12% polyacrylamide gel was used for the second dimension. Upon completion of electrophoresis the polypeptides were stained with Coomassie blue. Several spots were visualized, and the cores of two of them were manually excised before being desiccated for 30 min in a Speed Vac and washed with a solution of 50% acetonitrile, 50 mM ammonium hydrogenocarbonate (pH 8). Trypsinolysis was performed by using porcine trypsin (0.5 µg; Promega) for 16 to 18 h at 37°C. The peptides obtained were analyzed by using MALDI-TOF (Voyager, DE super STR; Applied Biosystems).

Five adult female BALB/c mice (5 weeks old; Iffa-Credo, L'Arbresle, France) were immunized intranasally with 10 µg of VLPs on days 0 and 14 and with 10 µg of the mucosal adjuvant Escherichia coli heat-labile toxin (LT) as previously reported (7). Control mice received phosphate-buffered saline (PBS) with LT. Blood and fecal samples were collected from each mouse on days 0 and 35.

Serum samples (n = 1,078; 55.9% female, 44.1% male; age range, 2 months to 96 years) were collected at the Laboratory of Virology (Dijon's Hospital) between February 2000 and June 2001. There was no association between collection of the samples and the presence or absence of known recent gastrointestinal disease.

Enzyme-linked immunosorbent assay for detecting Dijon171/96 virus-specific serum immunoglobulin G (IgG) and fecal IgA antibodies in mice sera was carried out with VLPs as antigen (100 ng in each well) as previously described for rotavirus VLPs (7). For human sera, 1:100 dilutions were used and wells were also coated without VLP (100 µl of PBS). Specific antibodies were revealed by using 100 µl of a 1:2,000 dilution of peroxidase-labeled goat anti-human IgG (Bio-Rad, Marnes-la-Coquette, France). The positive threshold was at least twofold the optical density of the serum tested without VLP (cutoff value of >0.2).

The purified capsid protein was resolved by a SDS-10% PAGE in denaturing (samples were boiled for 1 min before SDS-PAGE) or nondenaturing conditions, transferred to a nitrocellulose membrane by using a Bio-Rad Transblot apparatus, and analyzed by protein immunoblotting. Human and mouse sera diluted 1:100 in PBS were added and revealed by using goat anti-human (Bio-Rad) or anti-mouse (Biosys) IgG peroxidase.

Sf9 cells infected with the rDijon171/96 virus released into the supernatant a protein which migrated on a Coomassie blue-stained 10% polyacrylamide gel as a 55- to 59-kDa doublet after CsCl purification (Fig. 1A) as already reported (2, 18, 21). The 55-kDa protein might represent a possible cleavage product of the expressed protein as previously suggested (2, 21). The doublet was observed at a density of 1.30 g/cm³, and when this fraction was examined by electron micrograph, virus-like particles 38 nm in diameter were observed (Fig. 2). The protein was also present in the cell lysates, and a minor band with an apparent molecular mass of 35 kDa was also observed (Fig. 1B). Such a protein has been shown to be a C-terminal cleavage product of the capsid protein (12, 15) and has been reported to be mainly cell associated (15, 18). A MALDI-TOF analysis of this minor band yielded 13 peptides which all matched, except one, with peptides of the C-terminal end of Dijon171/96 capsid protein. The latter matched with a peptide located at the N-terminal end of the protein (amino acids 54 to 69). Of course, the MALDI-TOF method does not allow for affirming that the peptide obtained from trypsinolysis is really the N-terminal peptide. Nevertheless, this result remains difficult to explain. Although this event may be unlikely after a two-dimensional electrophoresis, a contamination by another cleavage fragment of the capsid protein cannot be excluded. Of interest, monoclonal antibodies specific for epitopes localized on an N-terminal fragment of the protein (amino acids 31 to 70) have been shown unexpectedly by Yoda et al. to react with the small-molecular-weight proteins derived from fecal Norwalk strains (22). Such proteins have been shown also to be C-terminal fragments of the capsid protein.

fig.ommitted FIG. 1. Dijon171/96 recombinant capsid protein purified by centrifugation in CsCl gradient from culture supernatant (A) and from infected cells (B). Lanes 1, 2, 3, 5, 6, and 7 of panel A and lanes 1 to 6 of panel B show SDS-PAGE on a 10% polyacrylamide gel of CsCl fractions from the top to the bottom of the gradient. The proteins were visualized by staining with Coomassie blue. Densities of CsCl fractions 3 (A and B) and of fraction 2 (B) are 1.30 and 1.29 g/cm3, respectively. Lanes 4 and 7, molecular size markers. Black arrows indicate positive fractions, and the white arrow indicates the 35-kDa protein.

 

fig.ommitted   FIG. 2. Electron micrograph of CsCl-purified recombinant VLPs (from fraction 3 of Fig. 1A; buoyant density, 1.30g/cm3) stained with phosphotungstic acid, pH 7.0. Bar, 100 nm.

 
Mice immunized twice intranasally with purified Dijon171/96 VLPs developed specific high-titered serum IgG as well as fecal IgA, whereas control mice immunized intranasally with PBS did not develop any antibody response by day 35. For serum IgG, the geometric mean titers for immunization by VLP plus LT and by PBS plus LT were 5.49 and 1.70, respectively. For fecal IgA, the geometric mean titer for immunization by VLPs plus LT was 2.44, with a standard deviation of 0.33, and for immunization by PBS plus LT was 1.30. Analysis of Dijon171/96 VLPs by immunoblotting with sera from two children as well as with a murine serum showed an immunoreactivity only when VLPs were resolved on SDS-PAGE under nondenaturing conditions. Three bands with an apparent molecular mass between 80 and 97 kDa, probably representing oligomers as suggested by Hardy et al. (11), were recognized by the human and murine sera, whereas the 55x to 59-kDa doublet was not reactive (Fig. 3), suggesting that epitopes recognized by such antibodies are discontinuous. Finally, the minor 35-kDa protein observed on SDS-10% PAGE gels when VLPs were prepared from infected cells was not immunoreactive by immunoblotting (data not shown). Testing of a higher number of sera might have shown different patterns of reactivity; however, these results are consistent with previous results reported for murine antibodies by Hardy et al. (11), which showed that 7 out of 10 monoclonal antibodies induced against recombinant NV VLPs recognized discontinuous epitopes, whereas only 3 out of 10 recognized continuous epitopes. However, here we did not observe any reactivity of a 63-kDa oligomer of the minor protein as reported by Hardy et al. for recombinant NV (11).

fig.ommitted FIG. 3. Immunoreactivity of rDijon171/96 was analyzed by immunoblotting with a murine serum and serum from a 2-year-old child after migration on a SDS-10% PAGE gel in denaturing (lanes a) and nondenaturing (lanes b) conditions. For SDS-PAGE, arrows indicate the 55- to 59-kDa doublet and possible oligomers of the capsid protein located between 80 and 97 kDa. For immunoblotting, arrows indicate the immunoreactive bands. mw, molecular weight markers.

 
The prevalence of Dijon171/96 virus-specific antibodies by age group is shown in Table 1. The majority of sera from children younger than 6 months were reactive due to the presence of maternal antibodies. For children between 6 and 11 months of age, the percentage of reactive sera fell to 35.5% and increased during the next years of life to reach 75% in children aged 5 to 9 years. By these ages the percentage of positively reacting sera increased and reached a peak value of 89% in the third decade. Finally, it is of note that the seroprevalence had a decreasing trend for the groups of more than 50 years of age. Overall, 799 of 1,078 (74.1%) serum samples were positive for Dijon171/96 virus-specific antibodies. The use of antigens produced by recombinant baculoviruses in insect cells allowed for the examination of the seroprevalence to different prototype noroviruses in previous studies in industrialized countries (17). The strains used were mainly NV strain for genogroup I and Mexico strain for genogroup II, and the seroprevalence to these strains generally varied from 75 to 100%. One recent study conducted in Italy used Lordsdale virus (18) and reported a seroprevalence of 91%, higher than the seroprevalence to Dijon171/96 strain (Lordsdale genotype) we report here. Nevertheless, strains close to Dijon171/96 virus in polymerase region comparisons have been found to circulate in France since 1996.

fig.ommitted TABLE 1. Prevalence of IgG antibodies to Dijon171/96 virus detected by enzyme-linked immunosorbent assay in 1,078 patients in Dijon (France) from February 2000 to June 2001

 
Nucleotide sequence accession number. The GenBank accession number for ORF2 of Dijon171/96 virus is AF472623.

　

	ACKNOWLEDGMENTS

 
This work was supported by grants from the Conseil Regional de Bourgogne, the Ministère de la Recherche, and ECOS-Nord (no. CF99S01).

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