Characterisation of defective interfering RNAs in avian influenza A (H7N9) virus

Abstract

Defective interfering (DI) virus is a virus particle that consists of functional structural viral proteins but has lost its essential genetic information. The presence of DI-RNA has been reported in different subtypes of influenza virus, including H1N1, H3N8, H7N7 and influenza B viruses. However, the mechanism of DI-RNA generation in influenza virus remains unclear. Since avian influenza A (H7N9) can cause severe illness in human, the existence of DI-RNA in H7N9-infected individuals is still unknown. By performing RT-PCR and Sanger sequencing, abundant DI-RNA production was observed in the nasopharyngeal aspirate (NPA) of H7N9-infected patients. To demonstrate the robustness of DI-RNA production in avian influenza A (H7N9) virus, the H7N9 reference strain, A/Anhui/1/2013 (AH1), was used to infect tissue-culture cells and mice. When comparing with a H1N1 laboratory strain (A/WSN/1933), the production of DI-RNA is higher in the AH1-infected NHBE cells. Single molecular real-time (SMRT) sequencing was applied to identify different DI-RNA species in H7N9-infected individuals. Identical PB1 DI-RNA species were observed in the NPAs of H7N9-infected patients. Moreover, long overlapping sequences (up to 23bp) were observed in the clinical specimens, indicating that similar sequences at the breakpoint sites may be a cause of DI-RNA production in H7N9 virus. Since the preliminary data demonstrated that the recombinant virus consisted of inconsistent influenza polymerase and NP segments (H5-3P) resulted in the abundant DI-RNA production. In this study, the H7-3P recombinant virus confirmed that inconsistent influenza polymerase and NP segments lead to the production of DI-RNA. Eight H7N9 strains were selected for future studies to further investigate the mechanism of DI-RNA generation in influenza virus. By performing amino acid alignments and constructing maximum likelihood phylogenetic trees, matching for the PB1 and NP segments was observed in most H7N9 viruses in the first three waves, and about half of the H7N9 viruses in the fifth wave. The eight selected strains were selected under the following criteria; (1) in the same wave and region with (2) similar PB2 and PA segment if possible. The production of DI-RNA in these eight selected strains would be investigated in the future studies.