Reverse genetics, the generation of viruses from full-length cDNA copies of viral genomes, is known as "infectious cloning" and is one of the most powerful genetic tools in modern virology. Recombinant DNA technology allows us to analyze and manipulate genomes at the molecular level. However, for non-retroviral RNA viruses, the genome does not enter the DNA stage during replication, and directly manipulating fragile RNA genes is a challenge. The genome of influenza viruses is segmented and negative-stranded, so genetic manipulation of influenza viruses is more demanding.
In order to serve as a functional template for initiating transcription and replication, influenza viral RNA (vRNA) must be transcribed into positive-sense mRNA by a polymerase complex, which includes three viral polymerases (PB2, PB1, and PA) and nuclear protein (NP). Influenza viruses have a genome consisting of eight RNA gene segments that must be expressed and packaged to complete the viral replication cycle.
The reverse genetic system of influenza virus was established by two different research groups in 1999, relying on intracellular RNA polymerase I (Pol I) to synthesize influenza virus RNA. RNA polymerase I is an abundant ribozyme that transcribes ribosomal RNA. RNA polymerase I initiates and terminates transcription at defined promoter and terminator sequences, and the resulting transcript contains no additional nucleotides at the 5' or 3' ends. Therefore, this enzyme is well suited to produce influenza virus vRNA in the nucleus. Although influenza virus rescue systems are constantly improving, the basic concept of using the RNA polymerase I system to synthesize vRNA remains the same.
At present, the de novo synthesis of influenza viruses is mainly achieved by transfecting cells with 8-12 plasmids. Among them, the 8-plasmid system is the most extensive in rescuing influenza A viruses. Eight plasmids contain promoters and terminators encoding RNA polymerase I, and cDNAs for each influenza virus segmented genome are contained between the promoters and terminators. The proteins of the 8-plasmid system are transcribed and translated by Pol II, and this system needs to express at least 4 proteins (PA, PB1, PB2 and NP). A general 8-plasmid system can transcribe 8 genes of influenza virus and translate 10 proteins of influenza virus. Translating complete influenza virus proteins can improve the efficiency of influenza virus rescue.
protocol:
Cloning 8-plasmid:
Use a vector containing human polⅠ sequence as the expression vector of the rescue system to construct an 8-plasmid rescue system. This vector is a bidirectional dual expression vector. The human pol I promoter and mouse pol I termination sequence are inserted in reverse between the pol II promoter (CMV) and the termination sequence (SV40). Eight cDNAs of influenza virus (including UTR and ORF) are inserted into the vector. After transfection into cells, pol I is responsible for synthesizing influenza virus single-stranded negative-strand RNA, and pol II is responsible for transcribing influenza virus positive-strand mRNA and subsequently translating influenza virus proteins. This plasmid is special because it has polyA tails before and after the inserted fragment. It cannot be sequenced with universal primers and needs to be sequenced with specific primers for each gene.
Rescue influenza A virus:
1. Inoculate HEK-293T into a 6-well plate and culture for at least 95% confluence for 24 hours.
Note: Since transfection will add a large amount of toxic transfection reagents, the cells need to be in good condition and the quantity required is large. Due to the species specificity of the human pol I promoter, only primate-derived cells (293T or Vero cells) with high transfection efficiency can be used for influenza virus packaging. Generally speaking, the rescue effect of 293T is stronger than that of Vero. Because MDCK cells support the growth of many influenza viruses and can be grown in the same culture medium as 293T cells, exclusively cultured 293T-MDCK cells (1:1) can be used to improve the rescue efficiency of certain influenza virus strains.
2. In DMEM medium (without FBS), add 1 μg of each plasmid, totaling 8 μg of 8 plasmids, mix with the transfection reagent, and let stand at room temperature for 15-30 minutes.
Note: Plasmid: liposome=1:3; Plasmid: PEI=1:4. DMEM can be replaced with Opti-MEM.
3. Replace the DMEM medium (containing FBS) of HEK-293T and slowly drop the plasmid-transfection reagent complex into the 6-well plate.
4. Culture the cells in a 37°C incubator containing 5% carbon dioxide.
Note: 35℃ environment is also acceptable.
5. 24 hours after transfection, add TPCK-treated trypsin with a final concentration of 1μg/ml and continue culturing.
Note: TPCK trypsin is used to cleave the HA protein of influenza virus, making the virus infectious. Some virus strains do not require this trypsin.
6. 48 hours or 72 hours after transfection, collect the HEK-293T cell supernatant. At this time, the influenza virus is in the supernatant, but the titer is extremely low.
Note: Repeated freezing and thawing of cells three times will help release influenza viruses in the cells.
7. Drop the collected supernatant into MDCK cells, and the influenza virus infects the MDCK cells for amplification.
Note: It is also necessary to add TPCK-treated trypsin with a final concentration of 1μg/ml. The culture of influenza virus in chicken embryos is very effective: inoculate into 9-11 day old SPF chicken embryos. Amnioallantoic fluid was harvested after 48 hours of incubation. The presence of infectious viruses can be confirmed by the hemagglutinin (HA) test.
8. 48 hours after infection, collect the MDCK cell supernatant and perform plaque assay to determine the virus titer.
Note: The generally measured titer is around 5x10^6Pfu/ml. If you want to increase the influenza virus titer, continue to infect MDCK with the virus for amplification.
9. The identity of the rescue virus should be confirmed by sequencing each of the 8 viral gene segments.
10. The virus liquid can be stored at 4℃ for several weeks. -80℃ can be stored for a long time.
References:
1. Liu Liqi. Preliminary study on the pathogenicity of HgN2 avian influenza virus and construction of reverse genetic system [D]. Chinese Center for Disease Control and Prevention, 2009.
2. Lee C W. Reverse genetics of influenza virus[M]//Animal Influenza Virus. Humana Press, New York, NY, 2014: 37-50.