Development of a novel live but defective interfering virus for anti-influenza therapy

Abstract

Defective interfering (DI) virus is a type of viral particle lacking parts of their genome essential for its replication cycle. With the defects in the genome, DI virus is unable to replicate on its own without the help of standard virus. First discovered in influenza, the presence of DI virus has then been detected in numerous different RNA viruses. It is believed that the erroneous activity of the RNA-dependent RNA polymerase gives rise to progeny DI viruses, which are then propagated upon infection at high multiplicity of infection (MOI). Interestingly, DI virus possess the ability to interfere with standard virus replication. The defective viral genome can compete with full-length viral genome for host machinery and viral assembly. The shorter defective viral genome is also a potent activator of innate immune response. The antiviral property of DI virus makes it a potential candidate for antiviral therapy. In influenza, defective viral genomes often occur at the polymerase segments PB2, PB1 and PA. Previous studies have shown that influenza virus bearing a single DI segment from PB2 confers protective effect in mice. However, to fully utilize the therapeutic potential of DI virus in a clinical setting, it is important to generate pure DI virus with minimal risk of reassortment events. Whether a pure influenza DI virus harboring more than one DI segment can be generated is yet to be determined. Our group has previously identified H7N9 as a potent inducer of DI species in all three of its polymerase segments. In this study, we sought to harness this property of H7N9 for the benefit of therapeutic purpose. The viral RNA of a recombinant virus with the backbone of H1N1 and polymerase complex of H7N9 were subjected to Single Molecule Real Time (SMRT) sequencing to identify the most abundantly expressed DI species. With the use of cell lines stably expressing PB2, a pure PB2 DI (H7-WSN-DI322) virus was generated by reverse genetics. Cell lines stably expressing both PB2 and PB1 were then developed to rescue a pure PB1 DI (H7-WSN-DI477) virus, from which compatible PB2 DI species were identified. The resultant 2DI virus (H7-WSN-DI477/597) was able to attenuate viral replication up to 10-fold in vitro. To generate DI viruses harboring an additional PA DI segment, a cell line with stable expression of PB2, PB1 and inducible expression of PA was generated to overcome the cytotoxic effects. A PA DI virus (H7-WSN-DI416) was successfully rescued with the use of this cell line. All in all, this study demonstrates that a pure DI virus harboring more than one DI segment can be generated and exhibit antiviral potential.