The H275Y Neuraminidase Mutation of the Pandemic A/H1N1 Influenza Virus Lengthens the Eclipse Phase and Reduces Viral Output of Infected Cells, Potentially Compromising Fitness in Ferrets

Abstract

The H275Y amino acid substitution of the neuraminidase gene is the most common mutation conferring oseltamivir resistance in the N1 subtype of the influenza virus. Using a mathematical model to analyze a set of in vitro experiments that allow for the full characterization of the viral replication cycle, we show that the primary effects of the H275Y substitution on the pandemic H1N1 (H1N1pdm09) strain are to lengthen the mean eclipse phase of infected cells (from 6.6 to 9.1 h) and decrease (by 7-fold) the viral burst size, i.e., the total number of virions produced per cell. We also find, however, that the infectious-unit-to-particle ratio of the H275Y mutant strain is 12-fold higher than that of the oseltamivir-susceptible strain (0.19 versus 0.016 per RNA copy). A parallel analysis of the H275Y mutation in the prior seasonal A/Brisbane/59/2007 background shows similar changes in the infection kinetic parameters, but in this background, the H275Y mutation also allows the mutant to infect cells five times more rapidly. Competitive mixed-strain infections in vitro, where the susceptible and resistant H1N1pdm09 strains must compete for cells, are characterized by higher viral production by the susceptible strain but suggest equivalent fractions of infected cells in the culture. In ferrets, however, the mutant strain appears to suffer a delay in its infection of the respiratory tract that allows the susceptible strain to dominate mixed-strain infections. In 2009, the World Health Organization (WHO) declared the first influenza pandemic of this century and described the virus (H1N1pdm09) as naturally resistant to adamantanes but susceptible to the neuraminidase (NA) inhibitors oseltamivir and zanamivir (10, 29). In the past 3 years, isolated cases of oseltamivir resistance in H1N1pdm09 strains have been reported-almost always associated with the H275Y mutation within the NA genebut the overall level of resistance has remained relatively low (42) at ϳ1% in the United States (47), Ͻ1% in Canada (40), ϳ2.5% in Europe (14, 26), and Ͻ1.6% worldwide (26). In the United States, the fraction of cases of resistance not associated with oseltamivir exposure increased significantly from 11% in (11,18). That dominance of an oseltamivir-resistant H275Y MUT strain was surprising at the time because it had been shown that the mutation usually compromised strain fitness (30). A return to widespread oseltamivir resistance, with a mutated H1N1pdm09 virus, could have significant public health consequences (19).Laboratory experiments have often been used to assess a particular influenza virus strain's phenotype in cell culture and animal models, particularly in relation to oseltamivir resistance. Prior to 2007, experiments demonstrated strongly attenuated growth of H275Y MUT strains of H1N1 in vitro (1, 21), a higher viral titer inoculum required for the infection of ferrets (21, 30), and generally less-pathogenic infections in ferrets, quantified by reduced fevers and smaller inflammatory cell counts in nasal washes (30). This mirrored...

Quantifying relative within-host replication fitness in influenza virus competition experiments

“…τ L ¼ 1=k) from 1 h to 24 h, consistent with previous estimates obtained when fitting models (with normal or lognormal delay distributions for L and I) to in vitro data Pinilla et al, 2012). δ…”

“…In order to produce more biologically accurate distributions for the latent and infected cell lifespans, the L and I compartments are each split into 20 stages, for both strain A and strain B (Petrie et al, 2013). The model is similar to that used by Pinilla et al (2012) to model competition between co-infecting strains in cell culture.…”

“…Previous studies have quantified the relative replication fitness of different pathogens using mathematical models (Marée et al, 2000;Wu et al, 2006;Hurt et al, 2010;Pinilla et al, 2012;Butler et al, 2014). A method introduced by Marée et al (2000)) quantified relative in vitro fitness via the ratio of the infected cell replication rates of each competing strain.…”

“…However, we neglected other possible causes of strain difference, such as strain-dependence in the duration of either the latent or productive phase of infected cells. and Pinilla et al (2012) quantified relative in vitro fitness by estimating several fundamental biological properties of each strain, gaining direct insight into the underlying cause of strain-dependent infection dynamics and fitness differences. Quantifying relative fitness in this way is preferable to estimating experiment-specific properties that change depending upon modifications to the infection system (e.g.…”

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

“…Researchers recently found that the H274Y NA substitution in H1N1pdm09 influenza A viruses reduces viral fitness after infection in ferrets, potentially by increasing the eclipse phase of viral growth (i.e., the time between infection and production of infectious virus) (78). In a genetic background without permissive mutations, reduced cell surface NA protein expression could increase the length of time necessary for infectious virus to bud and be released from an infected cell.…”