Spherical Influenza Viruses Have a Fitness Advantage in Embryonated Eggs, while Filament-Producing Strains Are Selected In Vivo

Abstract

Influenza viruses can take on two distinct morphologies: filamentous or spherical. While the functional significance of each virion type is unclear, filaments are generally observed in low-passage-number isolates, while an exclusively spherical morphology is seen in strains grown extensively in laboratory substrates. Previous studies have shown that filamentous morphology is lost upon passage in eggs. The fact that the filamentous morphology is maintained in nature but not in the laboratory suggests that filaments provide an advantage in the host that is not necessary for growth in laboratory substrates. To test this hypothesis and identify naturally occurring mutations that alter morphology, we examined the effect of serial adaptation in eggs, MDCK cells, and guinea pigs. Two filamentous strains, A/Netherlands/602/2009 (H1N1) and A/Georgia/M5081/2012 (H1N1), were passaged in eggs and MDCK cells. Conversely, the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) was passaged in guinea pigs. We found that although passage in eggs and MDCK cells can lead to a loss of filaments, an exclusively spherical morphology is not required for highly efficient growth in either substrate. We did, however, identify two point mutations in the matrix of egg passage 10 isolates that confer spherical morphology and increased growth in eggs. In contrast, serial passage in guinea pigs resulted in the selection of filament-forming variants. Sequencing revealed point mutations to the PR8 matrix that, when introduced individually, yielded filaments. These findings suggest a functional role for filaments in the infected host and expand the breadth of mutations known to affect influenza virus shape.

The M Segment of the 2009 Pandemic Influenza Virus Confers Increased Neuraminidase Activity, Filamentous Morphology, and Efficient Contact Transmissibility to A/Puerto Rico/8/1934-Based Reassortant Viruses

“…5C and D). Also consistent with published data on 2009 pandemic strains (34,56), the rNL602 virus was highly filamentous (Fig. 5E and F), particularly when imaged associated with a cell monolayer.…”

“…M2 is a transmembrane channel protein that is involved in (i) membrane scission during viral egress (23); (ii) acidification of the virion during viral entry, which leads to release of vRNPs from the M1 protein (24)(25)(26)(27); and (iii) preventing premature conformational change of specific hemagglutinin proteins in the Golgi complex, mediated through control of the pH within this organelle (24,25,27). Both M1 and M2 have been identified as determinants of influenza virion morphology (28)(29)(30)(31)(32)(33), and point mutations in either protein can convert an exclusively spherical influenza virus (approximately 200 nm diameter) to one that produces a mixture of spheres and filaments (which can be greater than 1 m in length) and vice versa (28)(29)(30)(31)(32)(33)(34). As clinical isolates of influenza A virus overwhelmingly include some percentage of filamentous virions and laboratory adaptation frequently results in the generation of strains with an exclusively spherical morphology (29,34), an understanding of the biological relevance of filamentous influenza A virus particles has long been sought.…”

“…Both M1 and M2 have been identified as determinants of influenza virion morphology (28)(29)(30)(31)(32)(33), and point mutations in either protein can convert an exclusively spherical influenza virus (approximately 200 nm diameter) to one that produces a mixture of spheres and filaments (which can be greater than 1 m in length) and vice versa (28)(29)(30)(31)(32)(33)(34). As clinical isolates of influenza A virus overwhelmingly include some percentage of filamentous virions and laboratory adaptation frequently results in the generation of strains with an exclusively spherical morphology (29,34), an understanding of the biological relevance of filamentous influenza A virus particles has long been sought.…”

Residue 41 of the Eurasian Avian-Like Swine Influenza A Virus Matrix Protein Modulates Virion Filament Length and Efficiency of Contact Transmission

“…Clinical isolates of influenza A virus typically include filamentous virions, whereas lab adaptation often produces strains that exhibit an exclusively spherical morphology (24,28,29). The biological relevance of filamentous influenza virions is currently unclear.…”

“…The biological relevance of filamentous influenza virions is currently unclear. Mutations arising in the M1 gene can convert an exclusively spherical influenza virus to one that produces a pleomorphic mixture of spheres and filaments, and vice versa (23)(24)(25)(26)(27)(28). Elleman and colleagues (24) reported that the morphologies of the lab-adapted strains A/PR/8/34 and A/WSN/33 differ from that of the human isolate, A/Victoria/3/75, and they mapped the phenotypic difference to matrix protein residues 41, 95, and 218.…”