Incorporation of the influenza A virus NA segment into virions does not require cognate non-coding sequences.

&lt;p&gt;For each influenza virus genome segment, the coding sequence is flanked by non-coding (NC) regions comprising shared, conserved sequences and specific, non-conserved sequences. The latter and adjacent parts of the coding sequence are involved in genome packaging, but the precise role of the non-conserved NC sequences is still unclear. The aim of this study is to better understand the role of the non-conserved non-coding sequences in the incorporation of the viral segments into virions. The NA-segment NC sequences were systematically replaced by those of the seven other segments. Recombinant viruses harbouring two segments with identical NC sequences were successfully rescued. Virus growth kinetics and serial passages were performed, and incorporation of the viral segments was tested by real-time RT-PCR. An initial virus growth deficiency correlated to a specific defect in NA segment incorporation. Upon serial passages, growth properties were restored. Sequencing revealed that the replacing 5&amp;#39;NC sequence length drove the type of mutations obtained. With sequences longer than the original, point mutations in the coding region with or without substitutions in the 3&amp;#39;NC region were detected. With shorter sequences, insertions were observed in the 5&amp;#39;NC region. Restoration of viral fitness was linked to restoration of the NA segment incorporation.&lt;/p&gt;</p

Seasonal Genetic Drift of Human Influenza A Virus Quasispecies Revealed by Deep Sequencing

&lt;p&gt;After a pandemic wave in 2009 following their introduction in the human population, the H1N1pdm09 viruses replaced the previously circulating, pre-pandemic H1N1 virus and, along with H3N2 viruses, are now responsible for the seasonal influenza type A epidemics. So far, the evolutionary potential of influenza viruses has been mainly documented by consensus sequencing data. However, like other RNA viruses, influenza A viruses exist as a population of diverse, albeit related, viruses, or quasispecies. Interest in this quasispecies nature has increased with the development of next generation sequencing (NGS) technologies that allow a more in-depth study of the genetic variability. NGS deep sequencing methodologies were applied to determine the whole genome genetic heterogeneity of the three categories of influenza A viruses that circulated in humans between 2007 and 2012 in France, directly from clinical respiratory specimens. Mutation frequencies and single nucleotide polymorphisms were used for comparisons to address the level of natural intrinsic heterogeneity of influenza A viruses. Clear differences in single nucleotide polymorphism profiles between seasons for a given subtype also revealed the constant genetic drift that human influenza A virus quasispecies undergo.&lt;/p&gt;</p

Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells.

&lt;p&gt;Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40 degrees C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature sensitivity of PB2-, NP- and M-RNA replication was also tested by quantitative real-time PCR. Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40 degrees C. From genetic analyses, it appears that temperature sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication.&lt;/p&gt;</p