Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets

Abstract

The acquisition of neuraminidase (NA) inhibitor resistance by H5N1 influenza viruses has serious clinical implications, as this class of drugs can be an essential component of pandemic control measures. The continuous evolution of the highly pathogenic H5N1 influenza viruses results in the emergence of natural NA gene variations whose impact on viral fitness and NA inhibitor susceptibility are poorly defined. We generated seven genetically stable recombinant clade 2.2 A/Turkey/15/06-like (H5N1) influenza viruses carrying NA mutations located either in the framework residues (E119A, H274Y, N294S) or in close proximity to the NA enzyme active site (V116A, I117V, K150N, Y252H). NA enzyme inhibition assays showed that NA mutations at positions 116, 117, 274, and 294 reduced susceptibility to oseltamivir carboxylate (IC(50)s increased 5- to 940-fold). Importantly, the E119A NA mutation (previously reported to confer resistance in the N2 NA subtype) was stable in the clade 2.2 H5N1 virus background and induced cross-resistance to oseltamivir carboxylate and zanamivir. We demonstrated that Y252H NA mutation contributed for decreased susceptibility of clade 2.2 H5N1 viruses to oseltamivir carboxylate as compared to clade 1 viruses. The enzyme kinetic parameters (V(max), K(m) and K(i)) of the avian-like N1 NA glycoproteins were highly consistent with their IC(50) values. None of the recombinant H5N1 viruses had attenuated virulence in ferrets inoculated with 10(6) EID(50) dose. Most infected ferrets showed mild clinical disease signs that differed in duration. However, H5N1 viruses carrying the E119A or the N294S NA mutation were lethal to 1 of 3 inoculated animals and were associated with significantly higher virus titers (P<0.01) and inflammation in the lungs compared to the wild-type virus. Our results suggest that highly pathogenic H5N1 variants carrying mutations within the NA active site that decrease susceptibility to NA inhibitors may possess increased virulence in mammalian hosts compared to drug-sensitive viruses. There is a need for novel anti-influenza drugs that target different virus/host factors and can limit the emergence of resistance.

Figure 1. Crystal structure of the A/Vietnam/1203/04 (H5N1) NA molecule (Protein Data Bank:2HTY).

Shown are 11 residues in or near the enzyme’s active site (black) that were substituted in this study. Residues shown in purple are positions where a single amino acid substitution affects the stability of the recombinant A/Turkey/15/06-like (H5N1) influenza virus.

Figure 2. NA enzyme kinetics of the recombinant A/Turkey/15/06-like (H5N1) viruses.

Substrate conversion velocity (V₀) of NA is shown as a function of substrate concentration. Fluorogenic MUNANA substrate was used at a final concentration of 0 to 2000 µM. The viruses were standardized to an equivalent dose of 10^7.5 PFU/ml. Fluorescence was measured every 92 sec for 45 min at 37°C, using excitation and emission wavelengths of 355 and 460 nm, respectively.

Figure 3. Patterns of clinical outcome in ferrets inoculated with the recombinant A/Turkey/15/06-like (H5N1) viruses.

(A) Mild, prolonged illness, (B) mild, brief illness, and (C) severe illness or (comparable to WT) caused by the indicated recombinant viruses. Shown are change in body temperature and weight, total number of inflammatory cells and protein concentrations in nasal washes, and virus titers in the upper respiratory tract. The horizontal lines show the mean inflammatory cell counts and protein concentrations in the nasal washes of uninoculated animals. Values are the mean ± s.d. for three ferrets. The mean s.d. of all data points for change in body temperature and weight was ∼±4.5%. *, P<0.05, **, P<0.01 compared to WT virus (one-way ANOVA).

Figure 4. Replication of recombinant WT, E119A, and N294S viruses in internal organs and histologic changes in the lungs morphology of ferrets infected with these H5N1 viruses.

(A) Virus titers were determined for the lungs (4 lobes tested separately), nasal turbinates, tracheas, small intestines, and livers of ferrets on day 4 post-inoculation. Values (log₁₀EID₅₀/gram tissue) are the mean ± s.d. for two ferrets, unless it is indicated that the virus was detected in one of two ferrets. *, P<0.05, **, P<0.01 compared to WT virus (one-way ANOVA). (B) The images shown are hematoxylin-and-eosin-stained sections of lung tissue from ferrets inoculated with the WT, E119A, and N294S viruses obtained on day 4 post-inoculation. Lung showing bronchiole (b) with epithelial necrosis, intraluminal debris, and inflammatory cells. Lung alveoli (a) of the WT-infected ferrets are lined with interstitial septa (arrows) and have mild to moderate inflammatory cell infiltrates. The lung alveolar architecture of the ferrets inoculated with the E119A mutant is lost due to necrosis of alveolar pneumocytes and the interstitial septa. Lung alveoli of the N294S-infected ferrets are filled with inflammatory cells and hyperplastic pneumocytes obscuring interstitial septa. Magnification, ×20.