Efficient generation of recombinant influenza A viruses employing a new approach to overcome the genetic instability of HA segments

Ahmed Mostafa; Pumaree Kanrai; Henning Petersen; Sherif Ibrahim; Silke Rautenschlein; Stephan Pleschka

doi:10.1371/journal.pone.0116917

Efficient generation of recombinant influenza A viruses employing a new approach to overcome the genetic instability of HA segments

PLoS One. 2015 Jan 23;10(1):e0116917. doi: 10.1371/journal.pone.0116917. eCollection 2015.

Authors

Ahmed Mostafa¹, Pumaree Kanrai², Henning Petersen³, Sherif Ibrahim⁴, Silke Rautenschlein³, Stephan Pleschka²

Affiliations

¹ Center of Scientific Excellence for Influenza Viruses, National Research Center (NRC), Cairo, Egypt.
² Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany.
³ Clinic for Poultry, University of Veterinary Medicine Hannover, Hannover, Germany.
⁴ Department of genetic engineering, Veterinary Serum and Vaccines Research Institute (VSVRI), Agricultural Research Center (ARC), Cairo, Egypt.

Abstract

Influenza A viruses (IAVs) are the most relevant and continual source of severe infectious respiratory complications in humans and different animal species, especially poultry. Therefore, an efficient vaccination that elicits protective and neutralizing antibodies against the viral hemagglutinin (HA) and neuraminidase (NA) is an important strategy to counter annual epidemics or occasional pandemics. With the help of plasmid-based reverse genetics technology, it is possible that IAV vaccine strains (IVVS) are rapidly generated. However, the genetic instability of some cloned HA-cDNAs after transformation into competent bacteria represents a major obstacle. Herein, we report efficient cloning strategies of different genetically volatile HA segments (H5- and H9-subtypes) employing either a newly constructed vector for reverse genetics (pMKPccdB) or by the use of the Escherichia coli strain HB101. Both approaches represent improved and generalizable strategies to establish functional reverse genetics systems preventing genetic changes to the cloned (HA) segments of IAV facilitating more efficient rescue of recombinant IAV for basic research and vaccine development.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cloning, Molecular / methods*
Dogs
Genetic Vectors / genetics
HEK293 Cells
Hemagglutinin Glycoproteins, Influenza Virus / genetics*
Humans
Influenza A virus / genetics*
Madin Darby Canine Kidney Cells
Mutagenesis, Site-Directed
Neuraminidase / genetics*
Recombinant Proteins / genetics
Viral Proteins / genetics
Viral Proteins / metabolism

Substances

Hemagglutinin Glycoproteins, Influenza Virus
Recombinant Proteins
Viral Proteins
Neuraminidase

Grants and funding

This work was supported in part by the FluResearchNet, “Molecular Signatures Determining Pathogenicity and Species Transmission of Influenza A Viruses”, funded by the German Federal Ministry of Education and Research (BMBF, grant 01 KI 1006E, to S. P. and grant, 01 KI 1006D to S. R.) and by a fellowship of the German-Egyptian Research Long-Term Scholarship “GERLS” program co-funded by the Egyptian government and the German Academic Exchange Service (DAAD, to A. M.). This work was also supported by a fellowship of the Egyptian Science and Technology Development Fund (STDF, to S. I.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.