Expression and self-assembly of Grimsby virus: antigenic distinction from Norwalk and Mexico viruses

Abstract

A cDNA obtained from Grimsby virus (GRV), a Norwalk-like virus, purified from a stool sample of a symptomatic adult associated with a gastroenteritis outbreak in the United Kingdom, was used to obtain the complete nucleotide sequence of the second open reading frame (ORF2). The ORF2 sequence of GRV predicts a capsid of 539 amino acids (aa) which exhibits aa identities of 96% to Lordsdale virus, 67% to Mexico virus (MXV), and 43% to Norwalk virus (NV). The GRV capsid protein was expressed in insects cells by using a recombinant baculovirus, and the resulting virus-like particles (VLPs) possessed a protein with an apparent molecular weight of 58,000. Hyperimmune antisera raised against purified GRV, MXV, and NV VLPs were tested in an indirect enzyme-linked immunosorbent assay (ELISA) against GRV, NV, and MXV VLPs, revealing that GRV is antigenically distinct from both NV and MXV. The antigenic specificity of the GRV-hyperimmune antiserum was confirmed in an antigen capture ELISA using GRV-, NV-, or MXV-containing fecal specimens. The expression of the GRV capsid protein has, for the first time, allowed the antigenic comparison of three distinct recombinant Norwalk-like viruses.

FIG. 1

Amino acid sequence alignments of the GRV, LRV, MXV, and NV putative capsid proteins. Alignments were generated by the Clustal method. The hypervariable region of the GRV sequence is underlined, and residues matching GRV exactly are indicated by dashes. GenBank accession numbers for the sequences used in the alignments are as follows: LRV, X86557; MXV, U22498; and NV, M87661.

FIG. 2

Coomassie blue-stained 10% polyacrylamide gel following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified rGRV, rNV, and rMXV capsid proteins. Preparations were boiled prior to electrophoresis.

FIG. 3

Electron micrograph of rGRV VLPs showing 38-nm-diameter VLPs. Bar, 100 nm.