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. 2001 Dec;39(12):4288-95.
doi: 10.1128/JCM.39.12.4288-4295.2001.

Characterization of capsid genes, expressed in the baculovirus system, of three new genetically distinct strains of "Norwalk-like viruses"

G Belliot  1 , J S Noel, J F Li, Y Seto, C D Humphrey, T Ando, R I Glass, S S Monroe
Affiliations

Characterization of capsid genes, expressed in the baculovirus system, of three new genetically distinct strains of "Norwalk-like viruses"

G Belliot et al. J Clin Microbiol. 2001 Dec.

Abstract

"Norwalk-like viruses" (NLVs), members of a newly defined genus of the family Caliciviridae, are the most common agents of outbreaks of gastroenteritis in the United States. Two features of NLVs have hindered the development of simple methods for detection and determination of serotype: their genetic diversity and their inability to grow in cell culture. To assess the immune responses of patients involved in outbreaks of gastroenteritis resulting from infection with NLVs, we previously used recombinant-expressed capsid antigens representing four different genetic clusters, but this panel proved insufficient for detection of an immune response in many patients. To extend and further refine this panel, we expressed in baculovirus the capsid genes of three additional genetically distinct viruses, Burwash Landing virus (BLV), White River virus (WRV), and Florida virus. All three expressed proteins assembled into virus-like particles (VLPs) that contained a full-length 64-kDa protein, but both the BLV and WRV VLPs also contained a 58-kDa protein that resulted from deletion of 39 amino acids at the amino terminus. The purified VLPs were used to measure the immune responses in 403 patients involved in 37 outbreaks of acute gastroenteritis. A majority of patients demonstrated a fourfold rise in the titer of immunoglobulin G to the antigen homologous to the outbreak strain, but most seroconverted in response to other genetically distinct antigens as well, suggesting no clear pattern of type-specific immune response. Further study of the antigenicity of the NLVs by use of VLPs should allow us to design new detection systems with either broader reactivity or better specificity and to define the optimum panel of antigens required for routine screening of patient sera.

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Figures

FIG. 1
FIG. 1
Electron micrograph of CsCl-purified VLPs from BLV construct at a magnification of ×265,085, after negative staining. Complete VLPs (white particles) are indicated by an arrow without a tail. Broken VLPs (dark particles) are indicated by a single-tailed arrow, and the 20- to 22-nm subunit particles are indicated by a double-tailed arrow.
FIG. 2
FIG. 2
(A) SDS-PAGE on a 10% polyacrylamide gel of 10 μl of CsCl-purified VLPs from TV, FV, BLV, and WRV baculovirus-expressed proteins. The 64- and 58-kDa proteins are indicated. Lane M, protein weight marker. (B) Amino acid sequence of the expressed proteins from panel A. The NV sequence with an NH2-terminal arm (the shaded sequence), involved in capsid stability, is indicated as a reference (28). The first methionines of the 64-kDa proteins are indicated; the first amino acids of the 58-kDa proteins found in the BLV and WRV preparations are boxed.
FIG. 2
FIG. 2
(A) SDS-PAGE on a 10% polyacrylamide gel of 10 μl of CsCl-purified VLPs from TV, FV, BLV, and WRV baculovirus-expressed proteins. The 64- and 58-kDa proteins are indicated. Lane M, protein weight marker. (B) Amino acid sequence of the expressed proteins from panel A. The NV sequence with an NH2-terminal arm (the shaded sequence), involved in capsid stability, is indicated as a reference (28). The first methionines of the 64-kDa proteins are indicated; the first amino acids of the 58-kDa proteins found in the BLV and WRV preparations are boxed.
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