title: Efficient generation of recombinant influenza A viruses employing a new approach to overcome the genetic instability of HA segments.
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.
authors.affiliation: Center of Scientific Excellence for Influenza Viruses, National Research Center (NRC), Cairo, Egypt.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Mostafa
authors.affiliation: Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Pumaree
authors.initials: P
authors.lastname: Kanrai
authors.affiliation: Clinic for Poultry, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Petersen
authors.affiliation: Department of genetic engineering, Veterinary Serum and Vaccines Research Institute (VSVRI), Agricultural Research Center (ARC), Cairo, Egypt.
authors.forename: Sherif
authors.initials: S
authors.lastname: Ibrahim
authors.affiliation: Clinic for Poultry, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Rautenschlein
authors.affiliation: Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Pleschka
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004285:Dogs; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D061985:Madin Darby Canine Kidney Cells; D016297:Mutagenesis, Site-Directed; D009439:Neuraminidase / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins; D014764:Viral Proteins; D009439:Neuraminidase