Alternative reverse genetics system for influenza viruses based on a synthesized swine 45S rRNA promoter

Abstract

We generated an alternative reverse genetics (RG) system based on a synthesized swine 45S rRNA promoter to rescue the H3N2 subtype swine influenza virus. All eight flanking segment cassettes of A/swine/ Henan/7/2010 (H3N2) were amplified with ambisense expression elements from RG plasmids. All segments were then recombined with the pHC2014 vector, which contained the synthesized swine 45S rRNA promoter (spol1) and its terminal sequence (t1) in a pcDNA3 backbone. As a result, we obtained a set of RG plasmids carrying the corresponding eight-segment cassettes. We efficiently generated the H3N2 virus after transfection into 293T/ PK15, PK15, and 293T cells. The efficiency of spol1-driven influenza virus rescue in PK15 cells was similar to that in 293T cells by titration using the human pol1 RG system. Our approach suggests that an alternative spol1-based RG system can produce influenza viruses.

Investigating Influenza Virus Polymerase Activity in Feline Cells Based on the Influenza Virus Minigenome Replication System Driven by the Feline RNA Polymerase I Promoter

“…Although the primary sequence of the RNA PolI promoter from different species is not well conserved, the sequences around its transcription initiation sites are relatively conserved (2,(4)(5)(6)(7). The common nucleotide sequences around the transcription initiation site of mammalian animals (-10 to +10 nt [the transcription initiation site of the RNA PolI promoter was defined as +1]) were queried in the five obtained contigs and hit the 340 to 359 nt position of Contig1209.2 and the 2411 to 2430 nt position of Contig1056.4.…”

H9N2 Viruses Isolated From Mammals Replicated in Mice at Higher Levels Than Avian-Origin Viruses

“…Reverse transcription was carried out using the Uni12 primer (5′-AGCAAAAGCAAGG-3′) and avian myeloblastosis virus (AMV) reverse transcriptase (Takara) following the manufacturer’s instructions and the cDNA products were stored at −80°C until use. The eight segments of influenza A virus were amplified by PCR using universal primers (Wang et al, 2017) and Phusion high-fidelity DNA polymerase (Vazyme, Inc., Nanjing, China). The PCR products were cloned into the pHW2000 vector using an in vitro recombination approach (Wang et al, 2017).…”

“…The eight segments of influenza A virus were amplified by PCR using universal primers (Wang et al, 2017) and Phusion high-fidelity DNA polymerase (Vazyme, Inc., Nanjing, China). The PCR products were cloned into the pHW2000 vector using an in vitro recombination approach (Wang et al, 2017). The cloned plasmids were confirmed by sequencing with the Sanger method (Genewiz, Inc., Suzhou, China) with primers: 5′-CGCAAATGGGCGGTAGGCGTG-3′ (CMV-Forward) and 5′-TAGAAGGCACAGTCGAGG-3′ (BGH-Reverse).…”

Influenza Reverse Genetics—Historical Perspective

“…However, it has now been shown that the human RNA polymerase I promoter allows influenza virus generation in canine MDCK cells (Suphaphiphat et al 2010), in African green monkey Vero (Neumann et al 2005) and COS-1 cells, and in swine kidney cells (Chen et al 2014). Moreover, additional influenza virus reverse genetics systems have now been established based on the RNA polymerase I promoter from (1) mice (Zhang and Curtiss 2015), with the mouse RNA polymerase I promoter also being functional in baby hamster kidney BHK21 and in Chinese hamster ovary CHO cells (Zhang and Curtiss 2015); (2) chickens (Massin et al 2005;Zhang et al 2009); (3) dogs (Wang and Duke 2007;Murakami et al 2008); (4) nonhuman primates (Song et al 2013); (5) horses (Lu et al 2016); (6) swine (Wang et al 2017); and (7) salmon (Toro-Ascuy and Martín 2017). In addition, the earlier-described T7 RNA polymerase-based reverse genetics system (de Wit et al 2007) is not species-specific and was used for influenza virus generation in hu-man, canine, and quail cells.…”