NA Inhibitor Susceptibility and NA Enzyme Kinetics of Recombinant H5N1 Viruses

An NA enzyme inhibition assay, a reliable phenotypic assay used to characterize the NA inhibitor susceptibility of influenza viruses, showed altered susceptibility in all recombinant H5N1 influenza viruses except that with K150N substitution. The susceptibility of V116A, I117V, and N294S viruses to oseltamivir carboxylate or zanamivir was moderately reduced (mean IC₅₀ increase, 5-63–fold and 3-33–fold, respectively) as compared to that of WT virus. Virus carrying the E119A mutation was moderately more resistant to oseltamivir carboxylate (35-fold increase in mean IC₅₀ value) and markedly more resistant to zanamivir (>1200-fold increase in mean IC₅₀ value) than was WT virus (Table 2). The H274Y mutant was much more resistant to oseltamivir carboxylate (mean IC₅₀ increase, >900-fold) and slightly less susceptible to zanamivir (mean IC₅₀ increase, 3-fold) than WT virus. In contrast, the Y252H NA change conferred increased susceptibility to oseltamivir carboxylate (mean IC₅₀ decrease, 22-fold) (Table 2).

To determine the effect of the NA mutations on viral functional properties, we characterized their kinetic NA enzymatic parameters, including their Michaelis-Menten constants (K_m), which reflect NA affinity for the MUNANA substrate, their inhibition constants (K_i) for oseltamivir carboxylate and zanamivir, and their relative NA enzymatic activity (V_max) (Table 2, Figure 2). NA proteins harboring the V116A, I117V, and E119A mutations exhibited significantly lower affinity for the substrate (mean K_m decrease, 4–8–fold) than did other NAs studied. The K_i values of all NA glycoproteins were consistent with their IC₅₀ values (Table 2), suggesting that the reduced susceptibility to both NA inhibitors could be caused by decreased affinity of their mutant NAs to the antiviral drugs.

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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.

We also determined NA enzymatic V_max values for each of the recombinant H5N1 viruses (Figure 2) and calculated their V_max ratios in relation to that of WT NA glycoprotein (Table 2). NA proteins with mutations at residues 117 and 252 had significantly higher enzymatic activity than WT NA (P<0.01). The other five NA mutations studied significantly reduced the NA activity of A/Turkey/15/06 (H5N1) influenza virus (all V_max ratios, 0.1) (Table 2, Figure 2). Thus, the eight recombinant H5N1 viruses differed in both their NA enzyme kinetics and their in vitro susceptibility to oseltamivir carboxylate and zanamivir.

Pathogenicity of Recombinant H5N1 Viruses in Ferrets

Limited information is available on the pathogenicity of H5N1 influenza viruses carrying various drug-resistant NA mutations in ferrets [23], although it can be an indicator of pathogenic potential of these viruses in humans. We inoculated ferrets with 10⁶ EID₅₀ of each virus and observed four different patterns of clinical outcome (Table 3, Figure 3). Recombinant WT virus caused mild clinical disease signs of moderate duration, similar to those caused by wild-type A/Turkey/15/06 (H5N1) strain [10].

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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).

Table 3

Pathogenicity of the Recombinant H5N1 Influenza Viruses in Ferrets.

Reverse-genetics virus	Change in oseltamivir resistancea	No. of surviving/total no.	Mean survival time ± SDb	Relative inactivity indexc	Mean serum antibody responsed
Mild, moderate duration
WT	1.0	3/3	>21.0	0.00	160
H274Y	941.5	3/3	>21.0	0.01	240
Mild, prolonged duration
V116A	7.2	3/3	>21.0	0.00	80
K150N	1.2	3/3	>21.0	0.02	80
Mild, short duration
I117V	5.1	3/3	>21.0	0.04	80
Y252H	0.1	3/3	>21.0	0.06	80
Severe illness
E119A	35.3	2/3	15.3±0.3*	0.29*	80
N294S	63.3	2/3	17.7±0.3*	0.46*	240

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^aMean fold-change in IC₅₀ compared to that of WT virus.

^bDetermined by the Kaplan-Meier method (Mantel-Haenszel test).

^cDetermined once daily for 14 days of observation based on the scoring system described in Methods and was calculated as the mean score per group of ferrets per observation (day) over the 14-day period. The relative inactivity index before inoculation was 0.

^dHemagglutination inhibition (HI) titers determined in ferret sera 21 days p.i. with A/Turkey/15/06 (H5N1) virus. Data are from groups of 2 (for E119A and N294S viruses) or 3 ferrets.

*P<0.05 compared to WT virus (one-way ANOVA).

Mild but prolonged illness (duration ~ 10 days, relative inactivity index, RII≈0.01) was seen in ferrets infected with mutant V116A and K150N viruses (Table 3). Despite their weight gain throughout the infection, the ferrets showed a mean peak temperature increase of ~ 1.0–1.3°C on day 8 post-inoculation (p.i.) (Figure 3A). On days 4, 6, and 8 p.i. the peak nasal inflammatory cell counts in nasal washes was significantly higher in groups of ferrets inoculated with V116A and K150N recombinants than in those inoculated with WT virus (P<0.01). Virus shedding started at 1 day p.i. for mutant viruses and continued until 6 days p.i. Interestingly, on day 1 p.i. virus titers in nasal washes were significantly lower for animals infected with recombinant V116A and K150N viruses than those in animals inoculated with WT virus (P<0.05), but remained significantly higher on day 6 p.i. (P<0.05) (Figure 3A).

Mild, short illness (duration ~ 5–6 days) was observed in ferrets inoculated with H5N1 viruses carrying I117V and Y252H NA mutations: only slight temperature elevation (mean peak increase, 0.5 to 1.2° C), mild clinical features (RII≈0.05) (Table 3) and minor weight changes were detected (Figure 3B). Cell counts remained at the same level in nasal washes of animals infected with either WT or I117V viruses, and they returned to normal levels in ferrets inoculated with Y252H at day 6 p.i. Comparison of the protein concentrations in the nasal washes showed no significant differences among the I117V, Y252H and WT viruses, suggesting that upper respiratory tract inflammation in these groups was comparable. Recombinant I117V and Y252H viruses replicated less efficiently in the upper respiratory tract than did the WT strain (P<0.05) (Figure 3B).

Virus with the H274Y NA mutation exhibited virulence comparable to that of recombinant WT virus (Table 3, Figure 3C). In contrast, inoculation of ferrets with recombinant E119A and N294S viruses caused markedly different results. Animals showed more pronounced clinical signs of disease, including a slightly greater RII (≈0.3–0.4), and one of three ferrets in each group was euthanized before the end of the experiment due to severe lethargy or excessive weight loss (Table 3). The body temperature elevation and nasal-wash inflammatory cell count were significantly higher in animals inoculated with the E119A mutant than in those inoculated with recombinant WT virus (P<0.01) (Figure 3C). The recovery of ferrets inoculated with the N294S NA mutant was delayed, and they regained no weight during the observation period.

To characterize in more details the disease caused by recombinant E119A and N294S viruses, we increased the number of animals from three to five in each of the pathogenicity groups and repeated inoculation of ferrets with 10⁶ EID₅₀ of the WT, E119A, and N294S viruses (Figure S1). Importantly, all clinical symptoms of disease were similar to those observed in the first experiment (Figure 3C), including a slight increase in RII for the E119A and N294S (0.3 and 0.4, respectively) and substantial weight loss (~15%) of N294S-infected ferrets. We observed that nasal-wash inflammatory cell counts were significantly higher in animals infected with mutant viruses than in those infected with WT (P<0.01) (Figure S1). Thus, in two independent experiments, the infection of ferrets with viruses carrying E119A or N294S NA mutations consistently caused more severe influenza disease than WT virus. Taken together, our results showed that changes in the framework residues of the NA enzyme active site can markedly affect the pathogenesis of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets.

Cells, Viruses and Compounds

Madin-Darby canine kidney (MDCK) and human embryonic kidney (293T) cells were obtained from the American Type Culture Collection and maintained as previously described [24].

Eight plasmids were constructed from the DNA sequences of the 8 gene segments of wild-type A/Turkey/15/06 (H5N1) virus for the reverse-genetics generation of recombinant wild-type virus. Recombinant virus was generated by DNA transfection of 293T cells [37], and the point mutations (Table 1, Figure 1) were inserted into the NA gene of wild-type virus by using a Quickchange site-directed mutagenesis kit (Stratagene) [24]. Stock viruses were prepared in MDCK cells at 37°C for 72 h and their entire HA and NA genes were sequenced to verify the presence of the mutations. The recombinant viruses were designated according to their NA mutations (Table 1, Figure 1). All experimental work with the H5N1 recombinant viruses was performed in a biosafety level 3+ laboratory approved for use by the U.S. Department of Agriculture and the U.S. Centers for Disease Control and Prevention.

The NA inhibitors oseltamivir carboxylate (oseltamivir) ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]-1-cyclohexene-1-carboxylic acid) and zanamivir (4-guanidino-Neu5Ac2en) were provided by Hoffmann-La Roche, Ltd.

Stability and Infectivity of Recombinant H5N1 Viruses

The genetic stability of the viruses was monitored by sequencing of the HA and NA genes after transfection and after three passages in MDCK cells at a MOI of 0.001 PFU/ml. If different subpopulations were identified, those viruses were considered unstable.

The infectivity of recombinant H5N1 viruses was determined in MDCK cells by plaque assay and expressed as log₁₀PFU/ml [9]. Briefly, confluent MDCK cells were incubated at 37°C for 1 h with 10-fold serial dilutions of virus. The cells were then washed and overlaid with minimal essential medium containing 0.3% bovine serum albumin and 0.9% Bacto agar and incubated at 37°C for 72 h. The plaques were stained with 0.1% crystal violet solution containing 10% formaldehyde, and virus yield was determined. Plaque diameter of any 10 plaques was measured by the Finescale® comparator (Los Angeles, CA, USA).

NA Enzyme Activity and Kinetics

Modified fluorometric assay was used to determine the NA activity of the recombinant H5N1 viruses [38]. We measured the NA enzyme kinetics at pH 6.5 with 33 mM 2-(N-Morpholino)ethanesulfonic acid hydrate (MES; Sigma-Aldrich), 4 mM CaCl₂, and fluorogenic substrate 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA; Sigma-Aldrich; final substrate concentration, 0–2000 µM). All H5N1 viruses were standardized to an equivalent dose of 10^7.5 PFU/ml. The reaction was conducted at 37°C in a total volume of 50 µl, and the fluorescence of released 4-methylumbelliferone was measured every 92 sec for 45 min in a Fluoroskan II instrument (Labsystems) using excitation and emission wavelengths of 355 and 460 nm, respectively. To measure the inhibitory effect of oseltamivir carboxylate or zanamivir on NA activity, H5N1 viruses were preincubated for 30 min at 37°C in the presence of various concentrations of the drugs (0.00005–5 µM). The kinetic parameters Michaelis-Menten constant (K_m), maximum velocity of substrate conversion (V_max), and inhibitory constant (K_i) of the NAs were calculated by fitting the data to the appropriate Michaelis-Menten equations by using nonlinear regression in the commercially available GraphPad Prism 4 software (GraphPad Software, La Jolla, CA).

NA Enzyme Inhibition Assay

Recombinant H5N1 viruses were standardized to equivalent NA activity and incubated for 30 min at 37°C with NA inhibitors at concentrations of 0.00005–100 µM with MUNANA (Sigma-Aldrich) as a substrate. After 1 h, the reaction was terminated by adding 14 mM NaOH and fluorescence was quantified in a Fluoroskan II (Labsystems) fluorometer. The concentration of NA inhibitor that reduced NA activity by 50% relative to a control mixture with no inhibitor (IC₅₀) was determined by plotting the dose-response curve of inhibition of NA activity as a function of the compound concentration. Values are the mean of 2–3 independent determinations.

Assessment of Virus Pathogenicity in Ferrets

Pathogenicity was tested in 4- to 5-month-old male ferrets obtained through the ferret breeding program at St. Jude Children’s Research Hospital or from Marshall Farms (North Rose, NY). All ferrets were seronegative for influenza A H1N1, H3N2, and H5N1, and for influenza B viruses. Groups of three ferrets were inoculated intranasally under light isoflurane anesthesia with 10⁶ EID₅₀ of virus in 1 ml sterile phosphate-buffered saline (PBS). Clinical signs of infection, relative inactivity index [35], weight, and temperature were recorded daily. Activity level was assessed by using the following scoring system: 0, alert and playful; 1, alert but playful when stimulated; 2, alert but not playful when stimulated; and 3, neither alert nor playful when stimulated. The relative inactivity index was calculated as the mean score per group of ferrets per observation (day) over the 14-day period. Animals that showed signs of severe disease (respiratory signs [labored breezing, sneezing, wheezing, and nasal discharge], febrility, lethargy, steady weight loss, and neurological signs [hind-limb paresis, ataxia, torticollis, and tremor] and >25% weight loss were euthanized. Body temperature was measured by subcutaneous implantable temperature transponders (Bio Medic Data Systems Inc.).

To monitor virus shedding, nasal washes were collected from ferrets on days 1, 2, 4, 6, and 8 post-challenge. Virus was titrated in 10-day old embryonated chicken eggs by injecting 0.1 ml of serial 10-fold dilutions of the sample (three eggs per dilution) and expressed as log₁₀EID₅₀/ml. Inflammatory cell counts and protein concentrations in nasal washes were determined as described previously [39]. Briefly, the nasal washes were centrifuged at 2,000 rpm for 10 min. The cell pellet was resuspended in PBS, and the cells were counted microscopically in a hemacytometer. The total number of inflammatory cells was calculated on the basis of the initial volume of nasal wash. The protein concentration in cell-free nasal washes was measured by using a protein reagent from Bio-Rad (Hercules).

Titration of Virus in Organs

Two animals inoculated with WT, E119A, or N294S viruses were euthanized by intracardiac injection of Euthanasia V solution on day 4 post-inoculation, and tissue samples (~0.5 g each) were collected from lungs (4 lobes tested separately), nasal turbinate, trachea, spleen, liver, and small intestine. Samples were homogenized in 1 ml sterile PBS with antibiotics, and the virus titer (log₁₀EID₅₀/gram tissue) was determined in 10-day old embryonated chicken eggs.

Histopathologic Analysis

Tissues (lung, nasal turbinate, trachea, spleen, liver, and small intestine) were collected at the time of necropsy, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Five-micrometer-thick sections were stained with hematoxylin and eosin and studied by light microscopy.

Serologic Tests

Serum samples were collected from ferrets 3 weeks after inoculation, treated with receptor-destroying enzyme, heat-inactivated at 56 °C for 30 min, and tested by hemagglutination inhibition (HI) assay with 0.5% packed chicken red blood cells by a standard method as described previously [40].

Virus Sequence Analysis

Viral RNAs were isolated from virus-containing cell culture fluid after transfection, after three passages in MDCK cells or from ferret nasal washes by using the RNeasy Mini kit (Qiagen). Samples were reverse-transcribed and analyzed by PCR using universal primers specific for the HA and NA gene segments, as described previously [41]. Sequencing was performed by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude. The DNA template was sequenced by using rhodamine or dRhodamine dye terminator cycle-sequencing Ready Reaction kits with AmpliTaq DNA polymerase FS (Perkin-Elmer) and synthetic oligonucleotides. Samples were analyzed in a Perkin-Elmer Applied Biosystems DNA sequencer (model 373 or 377). DNA sequences were completed and edited by using the Lasergene sequence analysis software package (DNASTAR).

We thank Klo Spelshouse for illustrations and Sharon Naron for excellent editorial assistance. The NA inhibitors oseltamivir carboxylate and zanamivir were provided by Hoffmann-La Roche, Ltd. (Basel, Switzerland).