Self-assembly of the recombinant capsid protein of a bovine norovirus (BoNV) into virus-like particles and evaluation of cross-reactivity of BoNV with human noroviruses

Abstract

None of the enteric caliciviruses except Po/Sapo/GIII/Cowden/80/US replicates in cell culture, which complicates efforts to develop control strategies or to study viral replication. To develop serological assays for bovine noroviruses (BoNVs) and to determine the cross-reactivity of BoNV with human noroviruses, we generated two recombinant baculoviruses, rCV186-OH and rJNCV, to express the capsid genes of Bo/CV186-OH/00/US (Norovirus genogroup III [GIII], genotype 2 [GIII/2]). rCV186-OH expressed the expected 57-kDa capsid protein, but rJNCV expressed a truncated capsid protein of 35 kDa. Sequence analysis of rJNCV identified a single nucleotide deletion in the P domain of the capsid gene, which introduced a stop codon at amino acid 323. The recombinant capsid protein produced by rCV186-OH but not that produced by rJNCV self-assembled into virus-like particles (VLPs) similar to native BoNV. An antibody-capture enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA (Ag-ELISA) detected serum antibody and antigen, respectively, from calves infected with Bo/CV186-OH/00/US but not antibodies or antigens to other enteric viruses. In other tests of the GIII/2 BoNV Ag-ELISA, no cross-reactivity was observed with VLPs from one GI and four GII human noroviruses and porcine sapovirus Cowden strain. Because, like human noroviruses, BoNVs do not grow in cell culture, the BoNV VLPs will be useful in the serological assays described for the detection of BoNV antibody and antigen. Consistent with the phylogenetic analysis of the capsid genes of bovine and human noroviruses (M. G. Han, J. R. Smiley, C. Thomas, and L. J. Saif, J. Clin. Microbiol. 42:5214-5224, 2004), the results suggest that GIII/2 BoNV does not share significant antigenic relationships with the five characterized human noroviruses tested.

FIG. 1.

Western blot analyses of cell lysates (A) and cell culture supernatants (B) of Sf9 cells infected with rCV186-OH and cell culture supernatants (C) infected with rJNCV. The cells were harvested every 2 days from 1 to 13 dpi. The cell lysates and supernatants of Sf9 cells infected with rCV186-OH and the supernatants of rJNCV-infected cells were concentrated by ultracentrifugation at 112,700 × g for 2 h. The cell lysates and supernatants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were detected by using bovine hyperimmune antiserum Bo9114. Cell lysates (A) and supernatants (B) of rCV186-OH infected cells: lane 1, molecular mass marker (numbers on the left are in kilodaltons); lanes 2 to 8, proteins harvested at 1, 3, 5, 7, 9, 11, and 13 dpi, respectively; lane 9, proteins harvested at 3 dpi from Sf9 cells infected with rJNCV; lane 10, uninfected mock Sf9 cell culture lysates (A) and supernatants (B) harvested at 7 days after culture. A recombinant protein of 35 kDa was detected from cell lysates and supernatants of Sf9 cells infected with rJNCV (lane 9). (C) Cell culture supernatants of the rJNCV-infected cells producing a 35-kDa truncated capsid protein: lane 1, molecular mass marker (numbers on the left are in kilodaltons); lanes 2 to 7, supernatants of rJNCV-infected Sf9 cells harvested at 1, 3, 5, 7, 9, and 11 dpi; lane 8, supernatants of rCV186-OH-infected Sf9 cells harvested at 3 dpi; lane 9, uninfected mock Sf9 cell culture supernatants.

FIG. 2.

Immune electron micrograph of BoNV VLPs. The rCV186-OH VLPs were purified by CsCl gradients from the cell culture supernatants of recombinant baculovirus-infected Sf9 cells. The VLPs were incubated with bovine hyperimmune antiserum Bo9114 against Bo/GIII/CV186-OH/00/US and visualized by negative staining with 3% phosphotungstic acid (pH 7.0). The insets show immune electron micrographs of the enlarged BoNV VLPs (left) and native BoNVs (right). Bars, 50 nm.

FIG. 3.

Serum IgG antibody responses and diarrhea and viral shedding in 1- to 2-week-old Gn calves orally inoculated with GIII bovine norovirus Bo/CV186-OH/00/US. Feces were scored by using a scale from 0 to 4 (where 0 is normal), with diarrhea considered a score ≥3 and indicated by a horizontal bar for the average duration (4.5 dpi) and a dotted line for maximum duration (5 dpi). Viral shedding detected by Ag-ELISA is shown by the horizontal bar for average duration (8.5 dpi) and a dotted line for maximum duration (10 dpi).