Influenza viruses belong to the orthomyxoviridae
family consisting of negative sense, single-stranded, RNA viruses with segmented genomes 
. Even with the availability of annual vaccines against human influenza infection, seasonal epidemics still result in nearly 500,000 deaths worldwide 
. Additionally, 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) 
. 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. Sequence analysis of its gene segments revealed that several reassortment steps led to the emergence of this virus, as it contains HA, NP, and NS genes from the classical swine lineage, NA and M from the Eurasian swine lineage, a human PB1 that was seeded from an avian virus in approximately 1968, and avian PA and PB2 genes 
. Six of the eight pH1N1 gene segments are shared with North American triple reassortant (TR) swine viruses that, despite their longtime establishment in the swine population, rarely infect humans and exhibit limited human-to-human transmission 
. 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.
Both the NA and M segments encode viral proteins with key roles in the influenza assembly, budding and release processes, which are required for efficient viral transmission. NA encodes the type II transmembrane protein, neuraminidase, which is found as a tetramer on the surface of the host-derived lipid envelope. It functions during the final stage of viral budding where it cleaves sialic acid containing receptors to allow for the release of progeny virions from infected cell surfaces 
. A recent study investigating the contribution of the Eurasian-origin NA and M segments to the enhanced spread of the pH1N1 strain reported a direct role for the novel NA protein in efficient transmission between ferrets 
. In this report, all naïve animals exposed to ferrets which had been infected intranasally with the pH1N1 isolate, A/California/07/09 (Rec pH1N1), tested positive for influenza by neutralization assays, indicating 100% transmission efficiency. However, upon replacement of Rec pH1N1 NA and M with segments derived from the TR lineage, transmission efficiency was reduced to 50%, similar to that observed by wild-type TR and Eurasian viruses. This observation suggests an important role for at least one of the Eurasian segments in enhanced viral transmission of Rec pH1N1. Furthermore, in this same study, NA originating from the Eurasian lineage was shown to exhibit higher neuraminidase activity in vitro
compared to NA with TR origin. Moreover, strains containing Eurasian origin NA with increased enzymatic activity released more viral particles, thereby proposing a mechanism by which the NA segment may be contributing to enhanced viral transmission of the pH1N1 virus.
The M segment of influenza encodes two viral proteins, matrix protein (M1) and the M2 ion channel. While M2 is essential for uncoating of the virus during entry, M1 is known to be the key component for both assembly and budding 
. Accumulation of M1 at the plasma membrane, and its interaction with the cytoplasmic tails of the viral surface proteins, are thought to initiate bud formation by inducing membrane curvature 
. Additionally, M1 interaction with both newly synthesized viral ribonucleoproteins (vRNPs) and the viral nuclear export protein (NEP) are known to be required for translocation of the viral genome from the host cell nucleus 
. These essential functions of M1 during the production of progeny virions support a role for the novel M segment in the enhanced transmission of the pH1N1 strain. Strengthening this hypothesis, Chou et al. recently showed that A/Puerto/Rico/8/34 (PR8) expressing the M segment from A/California/04/2009 (ACal-04/09) has increased transmission efficiency in vivo
using the guinea pig model 
. In this study, no viral transmission occurred between guinea pigs infected with wild-type PR8 and naïve animals, whereas 50% transmission efficiency was observed between guinea pigs infected with the PR8 recombinant expressing ACal-04/09 M. Though these data implicate a role for the pH1N1 M segment, specifically the M1 protein, in the spread of influenza, the exact mechanism and key residue(s) that determine efficient transmission remain unknown.
Specific M1 residues are also known to affect virion morphology. Influenza virions can range from 100 nm spheres to filamentous particles reaching several micrometers in length 
. Evidence for M1 influence on virion morphology was first provided in a study which showed that replacement of the M gene of A/WSN/33, a spherical producing strain, with that of the filamentous virus A/Udorn/72 resulted in filamentous virus morphology 
. Specific amino acids within the M1 protein were later found to be required for the production of filamentous particles, including residues 41, 95, 102, 204, and 218 
. Though it is known that influenza morphology affects virus production, its role in viral transmission is still unclear. Early reports showed that most influenza strains isolated from humans are predominantly filamentous and, upon continual passage in egg or tissue culture, adopt a more uniformly spherical morphology 
. This switch in morphology correlates with an increase in virus titer 
. Therefore, it is likely that the increased levels of virus production by spherical influenza strains results in more efficient viral transmission.
In this study, we characterized the morphology of pH1N1 isolates, and found that they are predominantly spherical in our cultured cells. This was in contrast to the filamentous phenotype we observed with the closely related, but poorly transmissible, TR swine viruses. In addition, we found that, unlike other strains, pH1N1 M1 by itself can efficiently induce virus-like particles from transfected cells. To evaluate the contribution of M1 mutations in virus morphology, we rescued various pH1N1 virus containing mutations at M1 residues different between pH1N1 and TR. Our results revealed that pH1N1 M1 residues 30, 207 and 209 are involved in regulation of virus morphology and enhanced viral spread in vitro. These data suggest that a few mutations present in pH1N1 contribute to morphological change and efficient transmission of influenza viruses.