Whole-genome, deep pyrosequencing analysis of a duck influenza A virus evolution in swine cells.

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Bourret, Vincent 
Croville, Guillaume 
Mariette, Jérôme 
Klopp, Christophe 
Bouchez, Olivier 

We studied the sub-population level evolution of a duck influenza A virus isolate during passage in swine tracheal cells. The complete genomes of the A/mallard/Netherlands/10-Nmkt/1999 strain and its swine cell-passaged descendent were analysed by 454 pyrosequencing with coverage depth ranging from several hundred to several thousand reads at any point. This allowed characterization of defined minority sub-populations of gene segments 2, 3, 4, 5, 7, and 8 present in the original isolate. These minority sub-populations ranged between 9.5% (for segment 2) and 46% (for segment 4) of their respective gene segments in the parental stock. They were likely contributed by one or more viruses circulating within the same area, at the same period and in the same or a sympatric host species. The minority sub-populations of segments 3, 4, and 5 became extinct upon viral passage in swine cells, whereas the minority sub-populations of segments 2, 7 and 8 completely replaced their majority counterparts. The swine cell-passaged virus was therefore a three-segment reassortant and also harboured point mutations in segments 3 and 4. The passaged virus was more homogenous than the parental stock, with only 17 minority single nucleotide polymorphisms present above 5% frequency across the whole genome. Though limited here to one sample, this deep sequencing approach highlights the evolutionary versatility of influenza viruses whereby they exploit their genetic diversity, predilection for mixed infection and reassortment to adapt to a new host environmental niche.

EMEM, Eagle’s Minimum Essential Medium, FCS, Influenza A virus, LB, Luria–Bertani, MID, Mixed infection, NPTr, Newborn Pig Trachea, Pyrosequencing, Reassortment, SD, SNP, Viral evolution, Within-host diversity, a.f.t., allele frequency threshold, base pairs, bp, dNTP, deoxyribonucleotides triphosphate, fœtal calf serum, multiplex identifier sequence, single nucleotide polymorphism, standard deviation, Animals, Animals, Wild, Cell Line, Coinfection, Ducks, Evolution, Molecular, High-Throughput Nucleotide Sequencing, Influenza A virus, Influenza in Birds, Polymorphism, Single Nucleotide, RNA, Viral, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Swine
Journal Title
Infect Genet Evol
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Elsevier BV
Biotechnology and Biological Sciences Research Council (BB/G00479X/1)
Biotechnology and Biological Sciences Research Council (BB/H014306/1)
This work was supported by a grant from DEFRA and HEFCE under the Veterinary Training and Research Initiative to the Cambridge Infectious Diseases Consortium (VB, LT), BBSRC grants BB/H014306/1 and BB/G00479X/1 (LT), and the French Ministry of Agriculture, INRA and the French Région Midi-Pyrénées (GC, J-LG, VB).