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

Abstract

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.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 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, 5Ј5068 -GGCTCCCAG AATTCTGAATG-3Ј 5089 , and reverse primer 5Ј-6702 CAAAG AGATCTCCAGCCATTA-3Ј 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. Puri...

Qualitative and Quantitative Analysis of the Binding of GII.4 Norovirus Variants onto Human Blood Group Antigens

“…The NoV strains used for this study were isolated from patients suffering from gastroenteritis in France (i.e., Dijon171 and E1057 strains were US95/96 and Hunter GII.4 variants, respectively), Egypt (i.e., Cairo7, Cairo1, and Cairo4 isolates belonged to the Yerseke, Den Haag, and Osaka GII.4 variants, respectively), and the United States (i.e., the MD145 strain was representative of the Bristol variant), as documented previously (15,20,21,34) (Table 1).…”

“…The Dijon171 recombinant baculovirus has been documented previously (34). The MD145 baculovirus was a kind gift of Kim Green (NIAID, NIH, Bethesda, MD).…”

Interaction of Single Viruslike Particles with Vesicles Containing Glycosphingolipids

“…The simplicity of the assay, which does not require labeling of the viral particles, combined with low material consumption, makes it attractive in pharmaceutics, where methods allowing for screening of new compounds inhibiting virus binding and spreading are highly desirable. The single viral particle sensitivity makes it also competitive with conventional quantitative polymerase-chain-reaction-based diagnostics [8].…”

“…Although the concept is generic, we focus in this work on the norovirus genogroup II.4 Dijon strain [8][9][10] and its interaction with membranes containing the glycosphingolipids (GSLs) H type 1 and Lewis b [5(b)]. Norovirus is a nonenveloped RNA virus, 38 nm in diameter, with a symmetric icosahedral capsid assembled from 180 copies of the capsid protein arranged into 30 hexagonal facets [11] ( Fig.…”

Norovirus GII.4 antibodies in the Portuguese population

“…Since no information on the genetic characterization of norovirus infections in Portugal was available, we selected GII.4 New Orleans antigen as this GII.4 variant emerged in 2009 and continued to cause outbreaks in 2012 [13]. Compared to data from other countries, such as France which reported a prevalence of 74.1% [14]; Italy, 91.2% [15]; and the UK, 99.5% [8], the 70% we found in the Portuguese samples was relatively low. This variance could reflect real differences in exposure levels of norovirus in different European countries, or may be the result of the fact that the VLPs used in our study were unable to detect cross-reactive antibodies against non-GII.4 norovirus strains including GI [16].…”