Abstract
The reverse genetics (RG) system of influenza A viruses is well established. However, the conventional sequence-dependent method for cloning influenza genome segments is time-consuming and requires multiple processes (eg. enzyme digestion and ligation) and exhibits low cloning efficiency compared to the sequence-independent cloning method. In this study, we improved influenza genome cloning into the pHW2000 vector for an RG system by incorporating a sequence-independent circular polymerase extension cloning (CPEC) approach which requires only 2 steps (reverse transcription and one-pot CPEC-PCR) and takes about 4 hours before the transformation. The specifically designed viral gene and vector primers used for CPEC-PCR have improved cloning efficiency ranging from 63.6 to 100% based on the results of gene-specific colony PCR which was additionally confirmed by enzyme digestion. We successfully cloned all genes from broad subtypes of influenza A viruses (H1-H12, N1-N9) and rescued by the RG system. Our results demonstrate that this method—one-Pot cloning for influenza A virus—was efficient in terms of required time and cloning rate. In conclusion, the novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses.
Highlights
The influenza A viruses (IAVs) which belong to the Orthomyxoviridae family, consist of eight segments[1]
Purified vectors were obtained through gel purification; the eluted vector DNA samples had a concentration of roughly 100~150 ng/μl acquired from 100 μL polymerase chain reaction (PCR) reactions for each gene-specific vector
In order to simplify the process and reduce the time required for cloning methods used to: (i) analyze the molecular characterization of influenza viruses and (ii) produce vaccines and recombinant viruses, we modified the traditional approach and cloned 8 influenza A virus gene segments into the vector using the primers described above
Summary
The influenza A viruses (IAVs) which belong to the Orthomyxoviridae family, consist of eight segments (ie, PB2, PB1, PA, HA, NP, NA, M, and NS)[1]. The recent development of a gene cloning method which relies on a mechanism similar to a polymerase chain reaction (PCR) for amplification of DNA sequences, the so-called “Circular Polymerase Extension Cloning” (CPEC), was developed as a simplified sequence-independent cloning technology based entirely on the polymerase extension mechanism[15]. It has notable advantages (eg, high cloning accuracy and efficiency) and uses www.nature.com/scientificreports/. The new strategy was evaluated and confirmed by cloning the eight influenza genes from a wide range of influenza subtypes (H1-H12 and N1-N9) and rescuing these clones using the RG system
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