Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

Abstract

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.

Coinfections of the Respiratory Tract: Viral Competition for Resources

“…The viral kinetics parameters for each respiratory virus are summerized in Table 1. Influenza kinetics parameters have been estimated before [23,25,27], and our parameter estimates lie within the range of previous estimates. Some of these parameters have also been estimated for RSV, although they are derived from in vivo patient data [28].…”

“…Our model predictions as well as the experimental data are shown in Fig 2. While our model predictions don't exactly match the experimental measurements, we see the same trend of increasing RSV viral load and decreasing IAV viral load. Given the inherent error in viral titer measurements [24] and the difficulty in reproducing experimental results due to lack of unit standardization [25], our model manages to reproduce the data fairly well.…”

(In)validating experimentally derived knowledge about influenza A defective interfering particles

“…Herein, because of our focus on influenza A virus, we have assumed both STV-only and STV + DIP-infected cells will have the same lifespan and thus exhibit the same CPE profile. This assumption is supported by both our prior work [11,27–29] and findings reported in [21], but is in contradiction with that reported in [37], all in the context of influenza A virus infections in the presence of DIPs. Nonetheless, for viruses where DIP co-infection is truly protective from CPE, use of the CPE as the endpoint in the B&C calculation will result in a valid, linear relationship only if the virus' co-infection window is of intermediate duration relative to its eclipse phase (as with STV yield reduction), but would not require that co-infected cells produce negligible amounts of STV (as is needed when using STV yield reduction).…”

Coinfection of semi-infectious particles can contribute substantially to influenza infection dynamics

“…Previously, mathematical models have been used to describe IAV viral load dynamics [12], estimate drug efficacies [13,14], and characterize the role of innate and adaptive immunity [15][16][17]. While some models have included 'non-infectious' virions [18][19][20], the roles that coinfection and complementation between SIPs play in regulating within-host IAV dynamics has not been studied. This knowledge is crucial to a quantitative understanding the extent of co-infection and reassortment during infection [11].…”