Influenza Virus Reassortment Occurs with High Frequency in the Absence of Segment Mismatch

Abstract

Reassortment is fundamental to the evolution of influenza viruses and plays a key role in the generation of epidemiologically significant strains. Previous studies indicate that reassortment is restricted by segment mismatch, arising from functional incompatibilities among components of two viruses. Additional factors that dictate the efficiency of reassortment remain poorly characterized. Thus, it is unclear what conditions are favorable for reassortment and therefore under what circumstances novel influenza A viruses might arise in nature. Herein, we describe a system for studying reassortment in the absence of segment mismatch and exploit this system to determine the baseline efficiency of reassortment and the effects of infection dose and timing. Silent mutations were introduced into A/Panama/2007/99 virus such that high-resolution melt analysis could be used to differentiate all eight segments of the wild-type and the silently mutated variant virus. The use of phenotypically identical parent viruses ensured that all progeny were equally fit, allowing reassortment to be measured without selection bias. Using this system, we found that reassortment occurred efficiently (88.4%) following high multiplicity infection, suggesting the process is not appreciably limited by intracellular compartmentalization. That co-infection is the major determinant of reassortment efficiency in the absence of segment mismatch was confirmed with the observation that the proportion of viruses with reassortant genotypes increased exponentially with the proportion of cells co-infected. The number of reassortants shed from co-infected guinea pigs was likewise dependent on dose. With 106 PFU inocula, 46%–86% of viruses isolated from guinea pigs were reassortants. The introduction of a delay between infections also had a strong impact on reassortment and allowed definition of time windows during which super-infection led to reassortment in culture and in vivo. Overall, our results indicate that reassortment between two like influenza viruses is efficient but also strongly dependent on dose and timing of the infections.

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

“…The probabilities of recovering any particular genotypes eight times within 21 or 23 reassortant viruses purely by chance are 2.7 × 10 −12 and 6.4 × 10 −12 , respectively, indicating that genetic reassortment between the EN and MO viruses is strongly biased. In comparison, a recent study of the genetic reassortment of influenza viruses differing only by two or three silent mutations in the middle of the gene segments demonstrated a high reassortment rate (88.4%), and the 123 different reassortant genotypes obtained from 241 purified viruses revealed no particular association between gene segments (37).…”

Spread and Persistence of Influenza A Viruses in Waterfowl Hosts in the North American Mississippi Migratory Flyway

“…The observations of gene dispersal and limited persistence of gene lineages among flyways are likely the result of countless introductions and subsequent removals of single gene lineages introduced by reassortment to AIVs in other flyways (19). The vast majority of reassortment events have negligible effects on AIV fitness and are presumably limited only by the rate at which multiple infections occur in host species (8). However, while it was not observed here, multiple studies have found persistence of entire AIV constellations across migratory seasons, suggesting the possible persistence of a virus in the environment until opportunistic exposure to some naive host species (62)(63)(64), although environmental conditions likely influence this persistence (65).…”

“…Influenza A virus is a single-stranded RNA virus of the order Orthomyxoviridae and contains eight separate RNA genomic segments that readily reassort with each other during coinfections to form ever-changing genomic constellations (8). In waterfowl, AIV infections are typically caused by low pathogenic (LP) avian-origin influenza A viruses (5,9; but see reference 10).…”

Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility

“…The importance of multiplicity reactivation during natural infection depends entirely on the prevalence of coinfection in vivo. Although the high frequency of reassortants generated during guinea pig infection demonstrates that coinfection occurs in vivo (4,26,27), its actual incidence is unknown.…”