Identification of the function and immunogenicity of virus-like particles of GII.4 Sydney 2012[P31] norovirus GZ2013-L10 strain in Guangzhou

Abstract:

[Background] Norovirus (NoV) is one of the main causes of global acute gastroenteritis. Among the viruses, GII.4 genotype persists through continuous mutation and is responsible for the majority of NoV infections. Particularly, since the emergence in 2012, GII.4 Sydney 2012[P31] variant has been prevalent all over the word. [Objective] To prepare virus-like particle (VLP) of GII.4 Sydney 2012[P31] NoV strain GZ2013-L10 in Guangzhou and systematically characterize its function and immunogenicity. [Methods] The ORF2 gene of GZ2013-L10 was amplified and cloned to construct the recombinant transposon vector pFastBac1-L10-ORF2. The vector was further transformed into Escherichia coli DH10Bac to develop the recombinant baculovirus plasmid, which was then transfected into insect cells sf9 for the expression of VLPs. The yielded VLPs were purified by ultracentrifugation. Finally, transmission electron microscopy, Western blotting, and receptor binding experiment were performed to characterize the VLPs. In addition, indirect enzyme-linked immunosorbent assay (ELISA) and test of the blocking of receptor binding were carried out verify the virus antiserum of the immunized mice. [Results] We successfully constructed the recombinant baculovirus plasmid Bacmid-L10-ORF2 and obtained VLPs. Electron microscopy demonstrated that the VLPs were about 30 nm in diameter. SDS-PAGE and Western blotting showed that the proteins were 58 kDa. Salivary receptor experiment results showed that the VLPs of GZ2013-L10 can bind to secretory salivary receptors such as A/B/O and porcine gastric mucosa protein (PGM) rather than non-secretory salivary receptors. Antiserum with a titer of 1.3×10⁵ was detected in the immunized mice. However, ELISA results showed no cross-immunoreactivity with capsid proteins of different NoV genotypes. In addition, the antiserum blocked the receptor binding of VLP of the same genotype but had no neutralizing effect on VLP of a different genotype (such as GII.8, GII.17, and GII.3). [Conclusion] VLP of GZ2013-L10 in Guangzhou and its antiserum were prepared and systematically characterized. The result can serve as a reference for elucidating the cause of the epidemic and developing vaccines.

Key words:Norovirus;virus-like particles;functional identification;immunogenicity;antiserum