Abstract:

Objective

In this study, the exogenous neocoronin gene (RBD gene) was inserted into the PA fragment of the polymerase gene of the temperature-sensitive cold-adapted virus strain A/Ann Arbor/6/1960(H2N2), a live attenuated vaccine of influenza A virus (IAV), and rewired the PA packaging signals of the H2N2 viruses to determine the boundary of the packaging signals of the PA genes, to construct H2N2 viral vectors that can be inserted into the exogenous genes based on the boundary sequence.

Methods

Using PCR to amplify 8 gene fragments of the A/Ann Arbor/6/1960(H2N2) virus, the RBD gene was inserted into the PA fragment in a reverse genetic manipulation system and the packaging signals of the PA genes were reassembled, that is, the packaging signal sequences of the polymerase genes were added or subtracted by 3 bases at a time. At the same time, in order to fully express the full length polymerase gene and the RBD gene, nonsense mutant packaging signals sequence, Porcine teschovirus-1 2A polypeptide sequence and exogenous gene sequence encoding influenza virus codon preference were inserted before packaging signal. Ligases were used to attach 8 gene fragments of the virus to the vector, and plasmids were constructed for each gene fragment. The recombinant virus was saved by transfection, and the MDCK cell was used to amplify the virus. The integrity of the virus gene was identified at the nucleic acid level to determine whether the packaging signal of influenza virus was damaged and the packaging signal boundary of influenza virus polymerase gene.

Results

A total of 11 PA constructs were created, encoding packaging signals ranging from 108～138 bp (excluding the stop codon). And the recombinant H2N2 viruses with packaging sequences of +12bp +15bp -6bp -9bp -12bp and -15bp were successfully rescued in the eight-plasmid IAV rescue system.

Conclusions

In this study, 6 recombinant influenza viruses are successfully rescued, that is, H2N2 vectors are successfully constructed that can insert exogenous genes. The packaging signal region of the recombinant H2N2 viruses is slightly different from that of PR8, suggesting that the location of packaging signals may differ between different subtypes.

Key words: H2N2 virus, packaging signals, novel coronavirus spike protein gene