Abstract
BackgroundReverse genetics is used in many laboratories around the world and enables the creation of tailor-made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of bacterial genomic sequences into the influenza gene of interest.MethodsHerein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation.ResultsHard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4–315/2014 (H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued.ConclusionThe LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be applied to clone any influenza gene segment using universal primers, which would help in rapid generation of influenza viruses and facilitate influenza research and vaccine development.
Highlights
Reverse genetics is used in many laboratories around the world and enables the creation of tailormade influenza viruses with a desired genotype or phenotype
The three polymerase genes (PB2, Polymerase Basic 1 (PB1) and Polymerase Acidic (PA)) contain same untranslated region (UTR) sequences, to improve the annealing efficiency of megaprimer with the bait plasmid, pHW2000 containing PA gene was taken as a bait plasmid (Fig. 1) and cloning PCR steps were performed
Transformation followed by growth of transformed bacteria at 37 °C resulted in colonies which showed Polymerase Basic 2 (PB2) and PB1 specific bands after screening with PB2 and PB1 specific primers
Summary
Reverse genetics is used in many laboratories around the world and enables the creation of tailormade influenza viruses with a desired genotype or phenotype. Reverse Genetics (RG) is the process of in vitro generation of live virus with synthetic or PCR amplified genes [1] This technique enables the creation of mutant influenza viruses of any desired genotype or phenotype. Reverse genetics as a tool has helped in studying the influenza host range [16, 17], transmission patterns [18] viral genome replication, pathogenicity and virulence [19,20,21] This technique has been implemented to develop influenza vaccines [22, 23] or recombinant influenza viruses harbouring reporter genes for studying virus egress and dissemination [24]
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