Enhanced propagation yield of influenza viruses for vaccine production through cellular microRNAs regulation

Abstract

Annual influenza vaccination aims to prevent influenza virus infection thus reduce the incidence of infection and disease in humans. Nowadays, Madin–Darby canine kidney (MDCK) cell culture-based system has been approved as an alternative for influenza vaccine production. Although cellular microRNAs may play an important role in controlling the replication of viruses, the interaction between human seasonal influenza viruses and host microRNAs has not been investigated in this permissive cell line. The primary objective of this study is to improve the yield of seasonal influenza virus (i.e. vaccine virus) production via manipulation of the host microRNAs. MDCK cells were infected with influenza A (pH1N1 or H3N2) or influenza B (Victoria or Yamagata) virus at MOI of 0.01. After collection at 6, 12, and 24 hours post-infection, microRNAs were subjected to massively parallel sequencing using MiSeq platform (Illumina). The profiles of microRNAs were validated by RT-qPCR. To evaluate the effect of candidate microRNAs on viral replication, MDCK cells were transfected with either microRNA inhibitors or microRNA over-expressing plasmids, followed by the infection of influenza viruses. The supernatants were collected at 48 hours post-infection, and the amount of virus was quantified by RT-qPCR and ELISA method. Moreover, in silico predicted microRNA binding sites were further verified by 3'-UTR reporter and dual luciferase assays. Furthermore, the effect of microRNA inhibitors on antigenic HA and NA sequences was also investigated. The results showed that four validated microRNAs including cfa-miR-340, cfa-miR-146b, cfa-miR-197, and cfa-miR-215 were most commonly upregulated upon infection with different seasonal influenza viruses. Among these microRNAs, cfa-miR-146b is a candidate target for microRNA inhibition to increase the production of A/pH1N1 and B/Yamagata viruses. Furthermore, cfa-miR-215 inhibition may be another good strategy for enhancing A/pH1N1 and B/Victoria viral production. On the other hand, the inhibition of cfa-miR-197 may be useful for the propagation of A/H3N2 virus. Moreover, the dual luciferase assay was used to evaluate microRNA binding sites. The results showed that the PB1 gene of A/pH1N1 virus is a direct target of cfa-miR-146b and cfa-miR-215, whereas the PB2 gene of A/H3N2 virus was silenced by cfa-miR-197. Whilst cfa-miR-146b targeted the PA gene of B/Yamagata, cfa-miR-215 silenced the PB1 gene of B/Victoria. Lastly, the sequencing results demonstrated that there was no change in antigenic HA and NA sequences between the viruses from the cells treated with microRNA inhibitors and the parental viruses. Taken together, it is demonstrated that microRNAs target viral genes in a strain-specific manner, leading to suppression of viral replication. Conversely, propagation of influenza viruses could be enhanced by the utilisation of microRNA inhibitors.