Influenza A virus (IAV) is an RNA virus with a segmented genome. These viral properties allow for the rapid evolution of IAV under selective pressure, due to mutation occurring from error-prone replication and the exchange of gene segments within a co-infected cell, termed reassortment. Both mutation and reassortment give rise to genetic diversity, but constraints shape their impact on viral evolution: just as most mutations are deleterious, most reassortment events result in genetic incompatibilities. The phenomenon of segment mismatch encompasses both RNA- and protein-based incompatibilities between co-infecting viruses and results in the production of progeny viruses with fitness defects. Segment mismatch is an important determining factor of the outcomes of mixed IAV infections and has been addressed in multiple risk assessment studies undertaken to date. However, due to the complexity of genetic interactions among the eight viral gene segments, our understanding of segment mismatch and its underlying mechanisms remain incomplete. Here, we summarize current knowledge regarding segment mismatch and discuss the implications of this phenomenon for IAV reassortment and diversity.
The reassortment of gene segments between influenza viruses increases genomic diversity and plays an important role in viral evolution. We have shown previously that this process is highly efficient within a coinfected cell and, given synchronous coinfection at moderate or high doses, can give rise to ϳ60 to 70% of progeny shed from an animal host. Conversely, reassortment in vivo can be rendered undetectable by lowering viral doses or extending the time between infections. One might also predict that seeding of transmitted viruses into different sites within the target tissue could limit subsequent reassortment. Given the potential for stochastic factors to restrict reassortment during natural infection, we sought to determine its efficiency in a host coinfected through transmission. Two scenarios were tested in a guinea pig model, using influenza A/Panama/2007/99 (H3N2) virus (wt) and a silently mutated variant (var) thereof as parental virus strains. In the first, coinfection was achieved by exposing a naive guinea pig to two cagemates, one infected with wt and the other with var virus. When such exposure led to coinfection, robust reassortment was typically seen, with 50 to 100% of isolates carrying reassortant genomes at one or more time points. In the second scenario, naive guinea pigs were exposed to a cagemate that had been coinoculated with wt and var viruses. Here, reassortment occurred in the coinoculated donor host, multiple variants were transmitted, and reassortants were prevalent in the recipient host. Together, these results demonstrate the immense potential for reassortment to generate viral diversity in nature. IMPORTANCE Influenza viruses evolve rapidly under selection due to the generation of viral diversity through two mechanisms. The first is the introduction of random errors into the genome by the viral polymerase, which occurs with a frequency of approximately 10؊5 errors/nucleotide replicated. The second is reassortment, or the exchange of gene segments between viruses. Reassortment is known to occur readily under well-controlled laboratory conditions, but its frequency in nature is not clear. Here, we tested the hypothesis that reassortment efficiency following coinfection through transmission would be reduced compared to that seen with coinoculation. Contrary to this hypothesis, our results indicate that coinfection achieved through transmission supports high levels of reassortment. These results suggest that reassortment is not exquisitely sensitive to stochastic effects associated with transmission and likely occurs in nature whenever a host is infected productively with more than one influenza A virus.T he segmented nature of the influenza virus genome allows for ready exchange of genetic material between two viruses that coinfect one cell (1). If the parental viruses differ in all eight gene segments, 256 different progeny viruses can be produced in a single reassortment event. Reassortment between two very distinct strains is typically associated with marked genotypic and ...
22implicated the PA gene segment as a major driver of this phenotype and quantification of viral 33RNA synthesis indicated that both replication and transcription were affected. These findings 34 indicate that multiple distinct mechanisms underlie IAV reliance on multiple infection and 35 underscore the importance of virus-virus interactions in IAV infection, evolution and emergence. 36Importantly, multiple infection with identical viral genomes can also alter infection 58 outcomes. Such cooperation was documented for VSV and HIV, where rates of transcription and 59 replication were enhanced with increasing multiplicity of infection (MOI) 23,24 . Similarly, faster 60 kinetics of virus production were seen at high MOI for poliovirus and IAV 19,25 . In these instances, 61 it is thought that increased copy number of infecting viral genomes provides a kinetic benefit 62 important in the race to establish infection before innate antiviral responses take hold. Indeed, it 63 has been suggested that multiple infection may be particularly relevant for facilitating viral growth 64 under adverse conditions, such as antiviral drug treatment 3,26 . 65For IAV, an important adverse condition to consider is that of a novel host environment. 66IAVs occupy a broad host range, including multiple species of wild waterfowl, poultry, swine, 67 humans and other mammals 27,28 . Host barriers to infection typically confine a given lineage to 68 circulation in one species or a small number of related species 29,30 . Spillovers occur occasionally, 69 however, and can seed novel lineages. When a novel IAV lineage is established in humans, the 70 result is a pandemic of major public health consequence 31,32 . The likelihood of successful cross-71 species transfer of IAV is determined largely by the presence, absence, and compatibility of host 72 factors on which the virus relies to complete its life cycle, and on the viruses' ability to overcome 73 antiviral defenses in the novel host [33][34][35] . 74Our objective herein was to assess the degree to which IAV relies on the delivery of 75 multiple viral genomes to a cell to ensure production of progeny. In particular, we sought to 76 determine whether this phenotype varies with host species. We therefore examined the 77 multiplicity dependence of one human and a panel of avian-origin viruses in multiple host systems. 78Results from all virus/cell combinations tested confirm prior reports that cells multiply-infected with 79 IAV produce more viral progeny than singly-infected cells. Importantly, however, the extent to 80 which viral progeny production is concentrated within the multiply-infected fraction of a cell 81 population varies greatly with virus-host context. Two poultry-adapted H9N2 viruses (A/guinea 82 fowl/HK/WF10/99 (GFHK99) and A/quail/HK/A28945/88 (QaHK88)) exhibit an acute dependence 83 on multiple infection in mammalian systems that is greatly diminished in natural host systems. 84This strong dependence on multiple infection is not seen for the human strain, influenza 85 ...
Influenza A virus (IAV) has a segmented genome, which (i) allows for exchange of gene segments in coinfected cells, termed reassortment, and (ii) necessitates a selective packaging mechanism to ensure incorporation of a complete set of segments into virus particles. Packaging signals serve as segment identifiers and enable segment-specific packaging. We have previously shown that packaging signals limit reassortment between heterologous IAV strains in a segment-dependent manner. Here, we evaluated the extent to which packaging signals prevent reassortment events that would raise concern for pandemic emergence. Specifically, we tested the compatibility of hemagglutinin (HA) packaging signals from H5N8 and H7N9 avian IAVs with a human seasonal H3N2 IAV. By evaluating reassortment outcomes, we demonstrate that HA segments carrying H5 or H7 packaging signals are significantly disfavored for incorporation into a human H3N2 virus in both cell culture and a guinea pig model. However, incorporation of the heterologous HAs was not excluded fully, and variants with heterologous HA packaging signals were detected at low levels in vivo, including in naïve contact animals. This work indicates that the likelihood of reassortment between human seasonal IAV and avian IAV is reduced by divergence in the RNA packaging signals of the HA segment. These findings offer important insight into the molecular mechanisms governing IAV emergence and inform efforts to estimate the risks posed by H7N9 and H5N8 subtype avian IAVs.
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