Construction and identification of influenza plasmid pool imparting high yields to candidate vaccine viruses in Vero cell at low temperature

Abstract

We generated plasmid pools for the rapid preparation of candidate vaccine strains, which could grow in the Vero cells at low temperature. Firstly, we cloned in the pHW2000 plasmid each of the eight gene segments (PB2, PB1, PA, hemagglutinin [HA], neuraminidase [NA], NS, NP, M) of two master donor strains (MDS), respectively, A/Yunnan/1/2005Vca(H3N2) and B/Yunnan/2/2005Vca(By), which had Vca phenotype (cold-adapted phenotype in Vero cells). Secondly, the similar operation was implemented with each of the HA, NA and NP segments of circulating strains with epidemic potential (parental strains). The virus rescue techniques were employed in this study, according to the homology rate of HA segments between MDS and parental strains. Then, we harvested amount of new Vca virus strains. By transmission electron microscope, it could observe new viruses' diameter and length were from 100 to 120 nm. Importantly, these reassortant viruses could get high-yield production in Vero cells at 25℃ from the beginning to the fourth generation, which was significantly differ from their original parental viruses. Additional, these production 16 new Vca strains could maintain enough antibody binding capacity and attenuation phenotype, which consisted with their MDS. So these plasmid pools constructed by mount of different influenza A and B virus gene fragments could present desired working performance and provide convenience and realization for more Vca reassortant virus as candidate vaccine strain if needing.

Keywords: Vero; high yield; influenza; plasmid pool; vaccine.

FIGURE 1

The results of 1% agarose gel electrophoresis with eight target typical gene plasmids by PCR detecting for these plasmid pools

FIGURE 2

Observation of virus particles by transmission electron microscope. The morphology was complete, and the background was clear. The envelope structure of the virus was clearly visible and showed typical spherical shape influenza virus particles. A, The new reassortant strain; (B) the parental strain

FIGURE 3

The virus yield change trend of 16 reassortment virus in Vero cell at 25°C by different types

FIGURE 4

The results of agar gel immunodiffusion test. The distribution of diameter for each virus matched human antibodies. All data were represented as means ± SD ($\overline{x}$ ± s) of three or more independent experiments