Increased virulence of neuraminidase inhibitor-resistant pandemic H1N1 virus in mice: potential emergence of drug-resistant and virulent variants

Abstract

Pandemic H1N1 2009 (A[H1N1]pdm09) variants associated with oseltamivir resistance have emerged with a histidine-to-tyrosine substitution in the neuraminidase(NA) at position 274 (H274Y). To determine whether the H274Y variant has increased virulence potential, A(H1N1)pdm09 virus, with or without the H274Y mutation, was adapted by serial lung-to-lung passages in mice. The mouse-adapted H274Y (maCA04H274Y) variants showed increased growth properties and virulence in vitro and in vivo while maintaining high NA inhibitor resistance. Interestingly, most maCA04H274Y and maCA04 viruses acquired common mutations in HA (S183P and D222G) and NP (D101G), while only maCA04H274Y viruses had consensus additional K153E mutation in the HA gene, suggesting a potential association with the H274Y substitution. Collectively, our findings highlight the potential emergence of A(H1N1)pdm09 drug-resistant variants with increased virulence and the need for rapid development of novel antiviral drugs.

Keywords: drug resistance; mouse adaptation; neuraminidase; pandemic influenza; virulence.

Figure 1. Comparison and characterization of virulence between mouse-adapted and parental viruses in BALB/c mice. Five independent serial lung-to-lung passages for each virus (CA04 and CA04_H274Y) were performed. After adaptation, 10^4.0 TCID₅₀ of the passaged viruses and their parental strains were administered intranasally (i.n.). Body weight loss and survival rate of the parental CA04 and CA04_H274Y with their corresponding mouse-adapted variants (maCA04_A, maCA04_B, maCA04_C, maCA04_D, and maCA04_H274Y_A, maCA04_H274Y_B, maCA04_H274Y_C, and maCA04_H274Y_D, respectively) were monitored in groups of 5 mice for 12 d p.i. (A–D). The number of passages is represented by ‘P’ followed by the name of the individually passaged viruses on the figure. For further characterization of lethal mouse-adapted oseltamivir-resistant variants, mouse-adapted viruses were plaque-purified and selected. Next, 10^3.0 TCID₅₀ of plaque-purified mouse-adapted CA04, mouse-adapted CA04_H274Y, and their parental viruses were administered to mice i.n. Body weight loss (E) and survival rate (F) were monitored daily for 12 d p.i. Groups of 12 mice were also given 10^3.0 TCID₅₀, and lung samples were collected 1, 3, 5, and 7 d p.i. to determine lung viral titers (3 heads/day) (G). Standard hematoxylin and eosin staining of lung tissues infected by CA04 (H), CA04_H274Y (I), mouse-adapted CA04 (J), and mouse-adapted CA04_H274Y (K) was examined 5 d p.i. (magnification × 100). *P < 0.05 (unpaired t test, two-tailed) of viral lung titers between wild-type CA04 and CA04_H274Y viruses and mouse-adapted CA04 and CA04_H274Y viruses. Error bar shown in (C, E, and G) represents standard error mean (SEM).