Abstract
In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus.
Highlights
Influenza viruses belong to the orthomyxoviridae family consisting of negative sense, single-stranded, RNA viruses with segmented genomes [1]
Because the two remaining gene segments, NA and M, were acquired from the Eurasian swine lineage, it is possible that these Eurasian swine genes and/or additional mutations created after the reassortment, contribute to the enhanced transmission of the pH1N1 virus among humans
Because efficient virion production has previously been shown to correlate with spherical rather than filamentous particle formation, we first determined the morphology of pH1N1 and triple reassortant (TR) viruses
Summary
Influenza viruses belong to the orthomyxoviridae family consisting of negative sense, single-stranded, RNA viruses with segmented genomes [1]. Influenza has been the cause of several global pandemics, most notably the 1918 Spanish flu which killed between 20 to 50 million people, and more recently the 2009 H1N1 swine-origin outbreak (pH1N1) [3]. This novel pH1N1 virus that emerged in April 2009, was declared a pandemic just two months later, already having spread to more than 70 countries. Because the two remaining gene segments, NA and M, were acquired from the Eurasian swine lineage, it is possible that these Eurasian swine genes and/or additional mutations created after the reassortment, contribute to the enhanced transmission of the pH1N1 virus among humans
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