Dynamics of Biologically Active Subpopulations of Influenza Virus: Plaque-Forming, Noninfectious Cell-Killing, and Defective Interfering Particles

Abstract

The dynamic changes in the temporal appearance and quantity of a new class of influenza virus, noninfectious cell-killing particles (niCKP), were compared to defective interfering particles (DIP). After a single high-multiplicity passage in MDCK cells of an egg-derived stock that lacked detectable niCKP or DIP, both classes of particles appeared in large numbers (>5 ؋ 10 8 /ml), and the plaque-forming particle (PFP) titer dropped ϳ60-fold. After two additional serial high-multiplicity passages the DIP remained relatively constant, the DIP/niCKP ratio reached 10:1, and the PFP had declined by about 10,000-fold. Together, the niCKP and DIP subpopulations constituted ca. 20% of the total hemagglutinating particle population in which these noninfectious biologically active particles (niBAP) were subsumed. DIP neither killed cells nor interfered with the cell-killing (apoptosis-inducing) activity of niCKP or PFP (infectious CKP), even though they blocked the replication of PFP. Relative to the UV-target of ϳ13,600 nucleotides (nt) for inactivation of PFP, the UV target for niCKP was ϳ2,400 nt, consistent with one of the polymerase subunit genes, and that for DIP was ϳ350 nt, consistent with the small DI-RNA responsible for DIP-mediated interference. Thus, niCKP and DIP are viewed as distinct particles with a propensity to form during infection at high multiplicities. These conditions are postulated to cause aberrations in the temporally regulated replication of virus and its packaging, leading to the production of niBAP. DIP have been implicated in the virulence of influenza virus, but the role of niCKP is yet unknown.

Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

“…Mixtures of rg-WT and either rg-H274Y or rg-R371K had increased mean IC 50 s only when the NAI-resistant population comprised at least 50% of the mixture. The oseltamivir-resistant phenotype was the most difficult to detect for rg-E119A, for which an increase in the mean IC 50 was detected only when it comprised at least 80% of the viral mixture.…”

“…Defective interfering (DI) or other noninfectious influenza virus particles can be produced during the course of infection and can be detected by deep sequencing (50)(51)(52). To independently quantify the proportions of NAI-susceptible and -resistant influenza B viruses after coinfection of NHBE cells and to confirm the results of deep sequencing, we performed high-resolution melt (HRM) analysis (42) on individually purified plaques from 3 pairs of mixtures (rg-WT and either rg-E119A, rg-H274Y, or rg-R371K; example melt curves are shown in Fig.…”

High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles

“…Such an understanding will also facilitate the engineering of DIPs for antiviral strategies and vaccines, as well as enable their control in bioprocess applications. Past studies have used coinfected cell cultures with DIPs and infectious virus to deduce DIP concentrations and elucidate their effects on virus and DIP production (2,29,(32)(33)(34)(35)(36)(37)(38). Vesicular stomatitis virus (VSV), grown on diverse cell cultures, has served for more than 50 years as a model for the study of DIPs (5,39).…”

“…The effects of DIP dose on virus yield have been elucidated (2,29,32,33,44) and have served as the basis for quantifying interference activity, assuming that individual DIPs are able to completely shut down virus production in coinfected cells (2,32,46). To date, however, relatively little has been revealed about how DIPs affect the intracellular steps that precede release of virus and DIP particles from coinfected cells.…”

Biological Activities of 'Noninfectious' Influenza A Virus Particles

“…Measuring the infectivity of IAV populations by these methods fails to account for the vast bulk of physical virions, as determined by hemagglutination end point or counting particles by electron microscopy (EM) [14,15]. In fact, particle-to-PFU ratios for IAV are typically greater than 10:1 [16,17]. This trait is not unique to IAV, as many other virus families also exhibit high particle-to-infectivity ratios.…”