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.
Highlights
Influenza viruses, belonging to the family Orthomyxoviridae, are divided according to the antigenic difference between their nucleoprotein (NP) and matrix 1 (M1) proteins into three genera; A, B or C [1]
The NAcDNAs of H9N2/SA and H5N1/Veterinary Serum and Vaccine Research Institute (VSVRI) were cloned into pCR2.1 for sequencing and further sub-cloning into the standard reverse genetics (RG) vector pHW2000
The insert DNA of these positive clones carrying the HA-cDNA of H9N2/SA and H5N1/VSVRI in pSMART-LC-Kan, confirmed by enzymatic digestion and sequencing, were subsequently sub-cloned into the RG vectors pHW2000 [44], pMPccdB [39] and pHH21 [14] and transformed into different competent bacterial strains (Top10, DH5-alpha, DH10B, XL1-Blue, Sure, Stbl2, Stbl3 and Stbl4), Figure 1
Summary
Influenza viruses, belonging to the family Orthomyxoviridae, are divided according to the antigenic difference between their nucleoprotein (NP) and matrix 1 (M1) proteins into three genera; A, B or C [1]. Out of these three genera, influenza A viruses (IAV) are the most likely to cause serious infections in different species including humans [2]. IAV is composed of eight negative sense genomic RNA segments within a lipid-bilayer envelope. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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