ArticleScientific reports2019
Development of a rapid, simple and efficient one-pot cloning method for a reverse genetics system of broad subtypes of influenza A virus.
Article in Scientific reports, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed, 8 citations in OpenAlex.
- Article
- Clade 2.3.4.4b highly pathogenic H5N1 influenza viruses from birds in China replicate effectively in bovine cells and pose potential public health risk.Emerging microbes & infections · 2025Article
- Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a.Immunity, inflammation and disease · 2024Article
- Multiple HA substitutions in highly pathogenic avian influenza H5Nx viruses contributed to the change in the NA subtype preference.Virulence · 2022Article
- Innate Immunity to Orthohantaviruses: Could Divergent Immune Interactions Explain Host-specific Disease Outcomes?Journal of molecular biology · 2022Review
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Authors and funding
10 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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