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Reduction of Neuraminidase Activity Exacerbates Disease in 2009 Pandemic Influenza Virus-Infected Mice Charlene Ranadheera,a,b Mable W. Hagan,a,b Anders Leung,a Brad Collignon,c Carissa Embury-Hyatt,c Darwyn Kobasaa,b

Todd Cutts,d Steven Theriault,d,e

Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canadaa; Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canadab; National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, Manitoba, Canadac; Applied Biosafety Research Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canadad; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canadae

ABSTRACT

During the first wave of the 2009 pandemic, caused by a H1N1 influenza virus (pH1N1) of swine origin, antivirals were the only form of therapeutic available to control the proliferation of disease until the conventional strain-matched vaccine was produced. Oseltamivir is an antiviral that inhibits the sialidase activity of the viral neuraminidase (NA) protein and was shown to be effective against pH1N1 viruses in ferrets. Furthermore, it was used in humans to treat infections during the pandemic and is still used for current infections without reported complication or exacerbation of illness. However, in an evaluation of the effectiveness of oseltamivir against pH1N1 infection, we unexpectedly observed an exacerbation of disease in virus-infected mice treated with oseltamivir, transforming an otherwise mild illness into one with high morbidity and mortality. In contrast, an identical treatment regime alleviated all signs of illness in mice infected with the pathogenic mouse-adapted virus A/WSN/33 (H1N1). The worsened clinical outcome with pH1N1 viruses occurred over a range of oseltamivir doses and treatment schedules and was directly linked to a reduction in NA enzymatic activity. Our results suggest that the suppression of NA activity with antivirals may exacerbate disease in a host-dependent manner by increasing replicative fitness in viruses that are not optimally adapted for replication in that host. IMPORTANCE

Here, we report that treatment of pH1N1-infected mice with oseltamivir enhanced disease progression, transforming a mild illness into a lethal infection. This raises a potential pitfall of using the mouse model for evaluation of the therapeutic efficacy of neuraminidase inhibitors. We show that antiviral efficacy determined in a single animal species may not represent treatment in humans and that caution should be used when interpreting the outcome. Furthermore, increased virulence due to oseltamivir treatment was the effect of a shift in the hemagglutinin (HA) and neuraminidase (NA) activity balance. This is the first study that has demonstrated that altering the HA/NA activity balance by reduction in NA activity can result in an increase in virulence in any animal model from nonpathogenic to lethal and the first to demonstrate a situation in which treatment with a NA activity inhibitor has an effect opposite to the intended therapeutic effect of ameliorating the infection.

I

n early 2009 a novel H1N1 subtype influenza virus crossed the species barrier from swine to humans to cause the first influenza pandemic since the H3N2 influenza pandemic in 1968. Initial cases of the pH1N1 virus were reported in March of 2009, and this virus has subsequently become established in the human population circulating as a seasonal strain contributing to a significant number of influenza-related deaths worldwide. Although it has been estimated that global fatalities due to the pH1N1 infection are upwards of more than 200,000, this virus typically causes a mild illness in otherwise healthy individuals and has demonstrated limited mortality in comparison to past pandemics (1, 2). Antivirals were the only form of intervention available to control the proliferation of disease during the first wave of the 2009 pandemic until the conventional strain-matched vaccine was produced. Presently, two classes of antivirals have been approved and marketed for use in humans to combat influenza virus infections, the M2-ion channel inhibitors and the neuraminidase (NA) inhibitors. The M2-ion channel inhibitors, which include amantadine and rimantadine, are potent inhibitors of influenza virus that block the migration of hydrogen ions into the virion and subsequent uncoating of the virus (3, 4), a necessary step in the viral life

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cycle (5). However, the rapid emergence of drug resistance has limited the use of this family of antivirals. On a global scale, upwards of 98% of circulating strains of influenza virus are currently resistant to this class of drug (6, 7). The NA inhibitors, which include oseltamivir and zanamivir (8, 9), act by inhibiting the release of progeny viruses from the host cell and subsequently preventing the spread of virus (10, 11). A variety of in vivo studies have demonstrated the clear efficacy of oseltamivir against a variety of seasonal and pandemic strains of influenza virus (12–17). Furthermore, oseltamivir has been shown to ease influenza-re-

Received 17 June 2016 Accepted 18 August 2016 Accepted manuscript posted online 24 August 2016 Citation Ranadheera C, Hagan MW, Leung A, Collignon B, Cutts T, Theriault S, Embury-Hyatt C, Kobasa D. 2016. Reduction of neuraminidase activity exacerbates disease in 2009 pandemic influenza virus-infected mice. J Virol 90:9931–9941. doi:10.1128/JVI.01188-16. Editor: D. S. Lyles, Wake Forest University Address correspondence to Darwyn Kobasa, [email protected]. © Crown copyright 2016.

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TABLE 1 Reference sequences of the 2009 pandemic H1N1 influenza virus isolates GenBank accession no. Isolate

HA

NA

M

NP

NS

PA

PB1

PB2

Mx10 Cal04 RV1532

FJ998208 FJ966082 GQ132146

FJ998214 FJ969517 GQ132164

FJ998211 FJ969513 GQ132174

FJ998217 FJ969512 GQ132169

FJ998220 FJ969514 GQ132182

FJ998223 FJ969515 GQ132151

FJ998226 FJ966080 GQ132140

FJ998206 FJ969516 GQ132156

lated clinical symptoms of seasonal strains (18–21). Many countries have now included the use of oseltamivir as a major component of their influenza pandemic preparedness strategies (22– 25), and it was used extensively in patients experiencing both mild and severe influenza-related symptoms during the 2009 pandemic (18, 26). In this study, we observed that oseltamivir treatment of the 2009 pandemic H1N1 (pH1N1) virus in mice had a deleterious outcome on survival. Furthermore, we demonstrated that treatment with oseltamivir and its subsequent reduction on NA function caused a change in the balance between the activities of the two viral surface glycoproteins, hemagglutinin (HA) and NA. Altering this balance unexpectedly turned an otherwise mild disease in mice into a lethal disease. MATERIALS AND METHODS Cells and viruses. MDCK cells were obtained from the American Type Culture Collection and maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum at 37°C with 5% CO2. A/InDRE/Mexico/4487/2009 (Mx10) was isolated from a patient during the early stages of the 2009 outbreak in Mexico and is very similar to A/California/04/2009 (Cal04). Influenza viruses were generated by reverse genetics, as previously described (27). Viruses were amplified in MDCK cells using MEM supplemented with 0.1% bovine serum albumin (MEM/ BSA) and TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone; 1 ␮g/ ml)-treated trypsin (MEM/BSA/trypsin). Viruses were subsequently sequenced to confirm that their nucleotide identity remained identical to that of the patient isolate. The GenBank accession numbers of the viruses used here were previously uploaded and are provided in Table 1. These references were used to confirm that the viruses used here were identical to the clinical isolate sequences. A/WSN/33 was kindly provided by Y. Kawaoka. Ethics statement. All animal procedures were performed as detailed in an Animal Use Document (H-11-009) approved by the Animal Care Committee of the Canadian Science Centre for Human and Animal Health according to the guidelines of the Canadian Council for Animal Care. Antiviral compounds. Commercial oseltamivir phosphate (Tamiflu) was acquired as capsules and dissolved in phosphate-buffered saline (PBS) for the oral treatment of mice. Oseltamivir carboxylate, the active metabolite of oseltamivir phosphate, was purchased from Toronto Research Chemical. Experimental animals and design. Female BALB/c mice, aged 5 to 6 weeks, were obtained from Charles River Laboratories. Mice were randomly assigned to each group, and all data obtained from each animal were used for analysis. Blinded studies were not conducted. In general, a minimum sample size of six mice was assigned to each treatment group to ensure adequate power to detect trends in survival and viral clearance. Each experiment consisted of at least three mice and was repeated at least once. Mice were treated by oral gavage with 100 ␮l of oseltamivir solution or PBS for five consecutive days. On day 0, the mice were anesthetized by inhalation of isoflurane, and 3 ⫻ 104 PFU of virus was delivered by intranasal (i.n.) inoculation in 50 ␮l of MEM/BSA. Clinical signs and weights were recorded daily, and group weights are presented. On sacrifice days, mice were anesthetized by inhalation of isoflurane, bled by cardiac punc-

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ture, and euthanized by an anesthetic overdose with isoflurane. Lung tissues were harvested and fixed in 10% buffered formalin for pathology or homogenized in MEM/BSA and clarified by centrifugation for viral titration on MDCK cells. Virus titrations. MDCK cells were seeded onto 96-well dishes 1 day prior to infection. Lung tissue was homogenized in 500 ␮l of MEM/BSA using a TissueLyser II (Qiagen). The homogenates were clarified by centrifugation at 10,000 ⫻ g for 10 min, and supernatants were serially diluted in MEM/BSA/trypsin. The cells were washed with MEM/BSA, and 100 ␮l of each virus dilution was added per well. The presence of a virusinduced cytopathic effect was read at 5 days postinfection (dpi) based on the appearance of rounded cells and/or destruction of the monolayer, and 50% tissue culture infective dose(s) (TCID50) were calculated according to the Spearman-Karber method (28). Each biological sample was tested in triplicate. The means and standard errors were plotted. Data were log transformed and analyzed by a two-way analysis of variance with no matching, followed by a Bonferroni posttest. If there were no surviving animals to assess at later time points and data for only one time point were collected, an unpaired two-tailed t test was used, and the equality of the variances was verified using an F test. Pathology. Lungs were harvested from Mx10-infected mice at 1, 2, 3, 6, and 8 dpi. For lung histopathological studies, samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (5 ␮m), and stained with hematoxylin and eosin for examination by light microscopy. For immunohistochemistry, paraffin tissue sections were quenched for 10 min in aqueous 3% H2O2 and then pretreated with proteinase K for 15 min. Slides were blocked for 30 min with Rodent Block M from MM HRP-polymer kit (Biocare Medical, USA) and then rinsed with Tris-buffered saline supplemented with 0.1% Tween. The primary antibody was a mouse monoclonal antibody specific for influenza A nucleoprotein (NP; F26NP9, produced in-house) and was used at a 1:10,000 dilution for 1 h. The slides were then incubated for 15 min with MM polymer-HRP (Biocare Medical) and reacted with the chromogen diaminobenzidine (DAB). The sections were then counterstained with Gill’s hematoxylin. Immunohistochemistry staining was assessed semiquantitatively by using a scoring system. Lung sections were given a bronchiolar (BR) score or an alveolar (AL) score based on the percentages of bronchiole or alveolar wall staining, respectively, in a given section as follows: 1⫹ ⫽ ⬍25%, 2⫹ ⫽ 25 to 50%, 3⫹ ⫽ 51 to 75%, and 4⫹ ⫽ 76 to 100%. RNA extraction. Lung, heart, spleen, kidney, and gut tissues were harvested from euthanized animals at 3, 6, and 8 dpi. Then, 30 mg of tissue was homogenized in 600 ␮l of RLT buffer (Qiagen) using a TissueLyser II. RNA was extracted using an RNeasy minikit (Qiagen) in accordance with the manufacturer’s instructions. Real-time reverse transcription-PCR. RNA extracted from the lung, heart, kidney, spleen, and gut were probed for the presence of pH1N1-HA RNA or WSN-matrix (M) RNA (29, 30) using a LightCycler 480 RNA master hydrolysis probe kit (Roche) and a LightCycler 480 (Roche) in accordance with the manufacturer’s instructions. Primer sequences are available on request. Neuraminidase enzyme activity assay. NA activity was measured using two different assay systems. Viruses were standardized to 106 PFU/ml and incubated at 37°C with a range of MUNANA [2=-(4-methylumbelliferyl)␣-D-N-acetylneuraminic acid] concentrations from 0 to 5,000 ␮M. Fluorescence was monitored every 90 s for 1 h. NA specificity

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was assessed using an Amplex Red neuraminidase assay (Invitrogen). Viruses were standardized to 106 PFU/ml and incubated at 37°C with a range of sialosides, 3=-sialyllactose, and 6=-sialyllactose substrates (Carbosynth, United Kingdom) at concentrations from 0 to 15,000 ␮M. The absorbance at 560 nm was monitored every 2 min for 5 h. The Michaelis constant (Km) and the maximum velocity (Vmax) were calculated using GraphPad Prism software by fitting the data to Michaelis-Menten equations using nonlinear regression. An extra sum of square F-test was used to compare individual values, and a P value was determined. Each assay was performed in triplicate, and each experiment was repeated three times. The means and the 95% confidence intervals (95% CIs) were tabulated. Virus sequencing. MDCK cells were seeded to confluence in a six-well plate 1 day prior to infection. The cells were washed with MEM/BSA immediately before use. Supernatants from lung homogenates were diluted in MEM/BSA and adsorbed onto cells for 1 h. The virus inoculum was removed, and an overlay containing 1% SeaPlaque agarose in MEM/ BSA/trypsin was added. The cells were incubated for 72 h at 37°C with 5% CO2. Individual plaques were isolated and expanded on MDCK cells. RNA was extracted from the cell supernatant using a QIAamp viral RNA minikit (Qiagen) according to the manufacturer’s recommendations. Each viral gene was amplified by reverse transcription-PCR and subsequently sequenced using the Sanger sequencing method to identify the presence of any point mutations. Virus growth with oseltamivir treatment. MDCK cells were seeded onto 12-well plates 24 h prior to use. The cells were infected with Mx10 or WSN at a multiplicity of infection (MOI) of 0.001 PFU diluted in MEM/ BSA. Fresh medium, i.e., MEM/BSA/trypsin containing 0 to 1,000 ␮M oseltamivir carboxylate, was exchanged for the virus inoculum after 1 h of adsorption. The supernatants were harvested at 48 h postinfection (hpi). Endpoint virus titrations were used, and TCID50 values were calculated as previously described. Each assay was performed in triplicate and repeated three times. The means and standard errors were plotted, followed by a variable-slope dose-response curve fitting using GraphPad Prism software (31). A 50% inhibitory concentration (IC50) was extrapolated from the fitted dose-response curve, and the means with 95% CIs were determined. Virus susceptibility to oseltamivir. A modified fluorometric assay, using substrate MUNANA (Sigma-Aldrich), was used to determine NA sensitivity to oseltamivir (32). Each assay was performed in triplicate, and each experiment was repeated three times. The results were plotted, followed by variable-slope dose-response curve fitting using GraphPad Prism software. An IC50 was extrapolated from the fitted dose-response curve and the means with 95% CIs were determined.

RESULTS

Treatment of influenza virus-infected mice with oseltamivir. BALB/c mice were used to assess the effectiveness of oseltamivir treatment of the 2009 pandemic H1N1 (pH1N1) influenza virus. Mice were given PBS or oseltamivir at 50 mg/kg/day orally for 5 days, beginning 1 day before infection with 3 ⫻ 104 PFU of either human wild-type isolate A/InDRE/Mexico/4487/2009 (Mx10; pH1N1) or human wild-type isolate A/California/04/2009 (Cal04; pH1N1) or the mouse-adapted highly pathogenic A/WSN/33 (WSN; H1N1) virus. The pH1N1 viruses used in this study, Mx10 and Cal04, are human viruses that were isolated early on during the 2009 pandemic in Mexico and California, respectively. The sequences of the viruses used here are identical to their respective human clinical isolates, as reported in GenBank (Table 1). PBStreated, Mx10-infected animals showed limited weight loss and no signs of disease, whereas similarly treated control mice infected with WSN presented severe clinical signs, together with substantial weight loss, requiring humane euthanasia by 5 dpi (Fig. 1A and Table 2). The converse was observed in animals treated with oseltamivir; Mx10-infected mice exhibited increased clinical signs of

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disease progression including severe weight loss (⬎25%), resulting in death or necessitating humane euthanasia by 8 dpi, whereas WSN-infected mice displayed no signs of disease or weight loss (Fig. 1A and Table 2), demonstrating successful treatment of the WSN infection. To ensure that these results were representative of other pH1N1 viruses and not an isolate-specific phenomenon, we also conducted the same experiments using the prototypic Cal04 isolate. In agreement with the Mx10 findings, mice infected with Cal04 and treated with oseltamivir demonstrated advanced clinical signs and weight loss requiring euthanasia by 8 dpi (Fig. 1A and Table 2), whereas untreated infected mice exhibited mild clinical signs of illness and experienced a maximum weight loss of only 5%. Furthermore, we also conducted preliminary experiments using another pH1N1 virus, A/Canada-AB/RV1532/2009, a patient isolate from Alberta, Canada, in early 2009, which also similarly demonstrated an aggravated disease progression requiring euthanasia when treated with oseltamivir (data not shown). Similar findings were seen in an expanded study with Mx10 when reduced doses of oseltamivir were used or the dosing schedule altered (Table 2), further supporting the association between the use of oseltamivir and the exacerbation of pH1N1-associated disease. Assessment of viral loads and pathology in the lungs of infected mice. Oseltamivir-treated mice infected with either Mx10 or WSN were serially sacrificed to determine virus loads in organs. Viral replication remained limited to the respiratory tract for all groups (Table 3). Mx10-infected mice treated with either PBS or oseltamivir had similar lung virus titers at 3 dpi of 9.5 ⫻ 106 PFU/g and 2.9 ⫻ 107 PFU/g, respectively (Fig. 1B). In both groups, the titers remained high until 6 dpi, dropping only by ⬃10-fold. Although both groups showed greater reduction by 8 dpi, lung titers remained ⬎300-fold higher in the oseltamivir-treated group than in the PBS-treated mice (Fig. 1B). We first observed increased weight loss and clinical signs, a finding indicative of a worsened outcome in oseltamivir-treated mice, at 4 and 6 dpi, respectively (Fig. 1A and Table 2). To illustrate the expected efficacy of oseltamivir treatment, we showed that WSN-infected mice treated with PBS had high virus titers at 3 dpi, whereas the oseltamivir-treated mice had a 178-fold-lower titers on the same day. By 8 dpi, WSN was no longer detected in treated animals, whereas all mocktreated animals had succumbed to the infection (Fig. 1B). Oseltamivir-treated mice infected with Mx10 were sacrificed at 1, 2, 3, 6, and 8 dpi to assess virus-induced lung pathology and antigen distribution. At 2 dpi, Mx10-infected, PBS-treated mice had predominantly necrotizing bronchiolitis lesions, whereas oseltamivir-treated mice additionally demonstrated interstitial inflammation and vasculitis (Fig. 1Ci and ii). By 6 dpi, necrotizing bronchiolitis was still present in both groups; however, the PBStreated mice exhibited evidence of regeneration, whereas the oseltamivir-treated mice had extensive pneumonitis and lesions continued to progress. At 8 dpi, only mild lesions were observed in the PBS-treated group (Fig. 1Ciii), while widespread moderate bronchiolitis and severe extensive interstitial inflammation and necrosis was evident in the oseltamivir-treated group (Fig. 1Civ). By 8 dpi, positive immunostaining was observed rarely in PBS-treated mice (Fig. 1Cv), whereas mice treated with oseltamivir showed widespread positive immunostaining (Fig. 1Cvi and Table 4). Overall, immunohistochemical analysis clearly indicated enhanced viral spread throughout the lungs by 6 dpi in mice treated with oseltamivir. Although a mea-

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FIG 1 Assessment of influenza virus-infected mice treated with oseltamivir. (a) Mice were infected with Mx10 (n ⫽ 24/treatment group) or WSN (n ⫽ 6/treatment group) virus strains, treated with either PBS or 50 mg/kg/day of oseltamivir for 5 days, and monitored daily, and the percent body weights and standard deviations were calculated. (b) Mx10-infected animals from each treatment group were sacrificed at 1 (n ⫽ 6), 2 (n ⫽ 6), 3 (n ⫽ 12), 6 (n ⫽ 12), and 8 (n ⫽ 12) dpi, and WSN-infected mice from each treatment group were sacrificed at 3, 6, and 8 dpi (n ⫽ 6). Lung virus titers were evaluated (means ⫾ the standard errors of the mean [SEM] were determined). *, No detectable virus; **, no survivors. (c) Assessment of lung histopathology and immunohistochemistry (n ⫽ 6/treatment group) in PBS or oseltamivir-treated mice infected with Mx10. (i) Necrotizing bronchiolitis (arrow) at 2 dpi; (ii) vasculitis/perivasculitis (arrow) and peribronchiolar inflammation (arrowhead) at 2 dpi; (iii) mild perivascular (arrowhead) and peribronchiolar inflammation (arrow) at 8 dpi; (iv) extensive bronchiolitis (arrows) and alveolitis with hemorrhage into alveolar spaces (*) at 8 dpi; (v) viral antigen in bronchiolar epithelial cells (arrowheads) at 8 dpi; (vi) viral antigen in bronchioles and alveolar walls at 8 dpi. Scale bars: panels i, ii, v, and vi, 50 ␮m; panels iii and iv, 100 ␮m.

surable difference in viral titers was apparent between treated and mock-treated animals by 8 dpi, the pathology results demonstrated a shift between increased disease progression in drug-treated animals and recovery in mock-treated animals occurring as early as 6 dpi.

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Taken together, these findings indicate that oseltamivir exacerbated the severity of infection with pH1N1 viruses, resulting in a lethal infection with a virus that otherwise caused mild illness in the BALB/c mouse model of infection.

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TABLE 2 Clinical signs and disease progression of influenza virus-infected BALB/c mice treated with oseltamivir Dose (mg/kg)a

Duration (days) of treatmentb

Clinical diseasec

Maximal % wt loss (dpi)

MTDd (days)

No. of mice tested

2 (OD) 10 (OD) 50 (OD) 50 (OD) 50 (OD) 25 (BID)

5 (⫺1 dpi) 5 (⫺1 dpi) 5 (⫺1 dpi) 5 (⫺1 dpi) 5 (3 hpi) 10 (⫺1 dpi) 5 (⫺1 dpi)

None Moderate Moderate Moderate Moderate Moderate Moderate

10 (7) 26 (7) 29 (7) 26 (8) 27 (7) 25 (6) 28 (7)

NL 7 7 8 7 6 7

24 6 8 24 6 6 6

PBS Oseltamivir

50 (OD)

5 (⫺1 dpi)

None Moderate

5 (4) 25 (8)

NL 8

8 8

PBS Oseltamivir

50 (OD)

5 (⫺1 dpi)

Severe None

26 (5) None

5 NL

6 6

Virus strain

Treatment

Mx10

PBS Oseltamivir

Cal04

WSN a

OD, once daily; BID, twice daily. The time before or after infection when treatment was initiated is indicated in parentheses. hpi, hours postinfection; dpi, days postinfection. Mice were evaluated according to clinical signs of illness, including ruffled fur, inappetence, level of activity, weight loss, and labored respiration. d MTD, mean time to death; NL, not lethal. Mice were infected i.n. with 3 % 104 PFU of virus and orally treated with PBS or oseltamivir phosphate. Disease progression was monitored for 18 days or until the animals reached a weight loss of ⬎25%, at which time they were humanely euthanized. b c

Sequence analysis of virus derived from infected lungs. Among the possible explanations for the observed outcome of treatment of pH1N1 infection in mice with oseltamivir is the selection and rapid expansion of pH1N1 variants, such as viruses conferring oseltamivir resistance, antibody escape, or mouse adaption. To evaluate this possibility, 25 plaque-purified viruses isolated from the lungs of oseltamivir-treated mice at 8 dpi were fully sequenced. We identified a range of point mutations found in the polymerase (PA, PB1, and PB2) and nonstructural (NS) genes (Table 5); however, there were no mutations conferring amino acid changes in the hemagglutinin (HA), NA, matrix (M), or nucleoprotein (NP) genes. The PB1 gene contained the most variations which appeared in multiple isolates, with one predominant mutation E51Q that occurred in 72% of the isolates (Table 5). Apart from this one mutation, the remaining mutations appeared to occur sporadically, and the general consensus sequence of the virus remained intact. None of these mutations have been associated with oseltamivir resistance or oseltamivir-related treatment (33, 34). Furthermore, these mutations have not been shown to be associated with mouse adaptation or viral adaptation to a new host (35–43). However, it is also important that the enhancement of pH1N1 infection occurred only with oseltamivir treatment and was not observed in mice treated with PBS, arguing against the

contribution of mouse adaptation to the observed virulence of the viruses. Evaluating NA activity and virus growth with oseltamivir treatment. The sensitivity of Mx10-NA to oseltamivir-mediated enzyme activity inhibition was similar to that of WSN-NA, yielding IC50s of 16.37 and 16.05 nM, respectively (Table 6). Oseltamivir carboxylate, the active metabolite of oseltamivir phosphate, inhibited the replication of both viruses in MDCK cells. A similar inhibition curve was observed for both WSN and Mx10, with no significant differences observed with respect to the effectiveness of oseltamivir at 48 hpi (Fig. 2). IC50s of 7.0 nM (95% CI ⫽ 0.0048 to 0.0103) and 7.8 nM (95% CI ⫽ 0.0034 to 0.0180) were obtained for WSN and Mx10, respectively, confirming similar sensitivities to oseltamivir in vitro. Correlation of NA activity with virulence. The sensitivity of pH1N1-NA enzymatic activity to oseltamivir suggested that the reduction in NA activity may have directly contributed to the enhancement of virulence in the mouse model. To evaluate this idea, recombinant Mx10 viruses containing NAs with lower enzymatic activity than Mx10-NA (Table 6) were generated by reverse genetics, including Mx10/NAH275Y, containing a mutation in the NA conferring oseltamivir resistance and decreased NA activity (44) (Table 6) and Mx10/U77NA, a virus with NA from

TABLE 3 Real-time RT-PCR detection of influenza virus in mouse organsa Detection of virus at various times (days) postinfection Lung

Heart

Spleen

Kidney

Gut

Virus strain

Treatment

Day 3

Day 6

Day 8

Day 3

Day 6

Day 8

Day 3

Day 6

Day 8

Day 3

Day 6

Day 8

Day 3

Day 6

Day 8

Mx10

PBS Oseltamivir

⫹ ⫹

⫹ ⫹

⫹ ⫹

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

– –

WSN

PBS Oseltamivir

⫹ ⫹

NA ⫹

NA –

– –

NA –

NA –

– –

NA –

NA –

– –

NA –

NA –

– –

NA –

NA –

a Mice were treated with PBS or oseltamivir phosphate at 50 mg/kg/day, starting 1 day prior to infection, for five consecutive days. Organs were harvested at 3, 6, or 8 dpi, and virus was detected by real-time RT-PCR. ⫹, positive detection of virus, –, virus was not detectable; NA, not available (the mice were previously euthanized, and the tissues were not available for assessment).

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4 4

3 3

4 4

1 3

2 4

1 2 Amplex Red 3= sialyllactose

a Mice were treated with PBS or oseltamivir phosphate at 50 mg/kg/day, starting 1 day prior to infection, for five consecutive days. Lungs were harvested 1, 2, 3, 6, or 8 dpi. Immunohistochemistry scores were based on the percentage of bronchioles (BR) or alveolar walls (AL) showing positive immunostaining in a given tissue section.

NA M NS

NP PA

PB1

PB2 a

C811T C1342T A972G NM NM C520T A249G G643A NM T1059G A1854G G279A T1344C G161C A101T T196G G204C A209C G347T A2146G A869G

Silent Silent Silent NM NM T170I T80A R211K NM N350K Silent Silent Silent E51Q S31C Silent S65T T67P K112N Silent S286G

4 4 8

4 4 8 4 4 8 8 72 20 56 12 24 4 4 4

Mice received oseltamivir at 50 mg/kg/day. NM, no mutation(s).

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Relative activity

Amplex Red 6= sialyllactose

Vmax (RFU/s)

HA

NA activityb

Gene segment Nucleotide mutation Amino acid mutation % of isolates

TABLE 6 Neuraminidase enzymatic properties and susceptibility to oseltamivira

TABLE 5 Sequence variation of lung virus isolates from Mx10-infected, oseltamivir-treated micea

Relative activity

A/USSR/90/1977 (H1N1). The Km values between viruses evaluated were comparable regardless of the substrate used, indicating similar affinities for the substrate (Table 6). However, as expected, the maximal NA activity levels (Vmax) of the Mx10/NAH275Y and Mx10/U77NA viruses were lower than those for Mx10 using the MUNANA substrate (Table 6). Furthermore, when the substrate specificity was assessed, similar trends in relative activity levels were observed between viruses when specific 3=- or 6=-sialyllactose substrates were used. These results confirm a lower NA activity for Mx10/NAH275Y and Mx10/U77NA for both ␣2,6-sialic acid (SA) and ␣2,3-SA substrates compared to Mx10 (Table 6). Untreated mice infected with Mx10/U77NA or Mx10/NAH275Y exhibited greater weight loss and more severe clinical signs compared to untreated-Mx10 infection (Fig. 3A), but similar to oseltamivirtreated Mx10-infected mice (Fig. 1A), requiring humane euthanasia at 7 and 8 dpi, respectively. High lung titers at 8 dpi, similar to what we found at 6 dpi, were observed for Mx10/NAH275Y, whereas the titers in Mx10-infected mice had dropped ⬎3,000fold between 6 and 8 dpi. Mx10/U77NA and Mx10/NAH275Y in untreated mice were similarly virulent compared to Mx10 in oseltamivir-treated mice, supporting the hypothesis that a reduction

ND, the IC50 was not calculated; NA, GraphPad Prism 5 could not calculate a P value because models with a shared IC50 are ambiguous. Mean values are presented for IC50, Km, and Vmax, and the 95% CI values are indicated in parentheses. The Km values are statistically not different between viruses (P ⬎ 0.05). The Vmax values are statistically different between viruses (P ⬍ 0.05). The relative activity is the ratio of the Vmax between the virus and Mx10. Note that the bottom two rows of the table indicate “F (DFn, DFd)” and “P” values, respectively.

1 1

b

4 2

a

1 1

0.0029 (0.0026–0.0032) 0.0015 (0.0014–0.0016) 0.0005 (0.0004–0.0005) 0.0014 (0.0013–0.0015) 79.92 (3, 76) ⬍0.0001

2 2

164.4 (51.99–276.8) 231.6 (164.3–298.8) 279.1 (175.7–382.6) 323.2 (232.1–414.3) 1.336 (3, 76) 0.2690

AL

1.00 0.64 0.24 0.52

BR

0.0025 (0.0020–0.0029) 0.0016 (0.0009–0.0022) 0.0006 (0.0005–0.0006) 0.0013 (0.0007–0.0018) 4.498 (3, 68) 0.0061

AL

2,013 (978.7–3,048) 2,360 (0.0–4,891) 3,660 (2,906–4,413) 1,359 (0.0–3,173) 0.2745 (3, 68) 0.8436

BR

1.00 0.42 0.50 0.74

AL

16.37 (14.87–18.02) 1433 (51.66–4016) ND 16.05 (12.87–20.01) NA NA

BR

Mx10 Mx10-NA H275Y Mx10-USSR NA WSN F (DFn, DFd) P

AL

Km (nM)

BR

Vmax (RFU/s)

AL

Km (nM)

BR

Vmax (RFU/s)

Day 8

Km (nM)

Day 6

Mean oseltamivir IC50 (nM)

Day 3

Virus strain or parameter

Mx10/PBS Mx10/oseltamivir

Day 2

MUNANA

Day 1

Virus strain/treatment

1.428 (1.19–1.67) 0.600 (0.51–0.69) 0.713 (0.54–0.88) 1.059 (0.93–1.19) 18.68 (3, 125) ⬍0.0001

Score at various times (days) postinfection

125.4 (46.79–203.90) 117.7 (52.23–183.10) 153.3 (22.03–284.60) 130.8 (72.41–189.20) 0.09542 (3, 125) 0.9624

Relative activity

TABLE 4 Immunohistochemistry scores of antigen prevalence from infected mouse lung tissuesa

1.00 0.52 0.17 0.48

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FIG 2 Sensitivity of influenza viruses to oseltamivir carboxylate in MDCK cells. MDCK cells were infected with Mx10 (A) and WSN (B) at an MOI of 0.001. After 1 h of adsorption, the virus inoculum was removed and replaced with medium containing 0 to 1,000 ␮M oseltamivir carboxylate (active form). Supernatants were harvested at 48 hpi, and the virus titer was determined on MDCK cells. Experiments were performed in triplicate, and results are reported as mean TCID50 values ⫾ the SEM.

in NA activity of the pH1N1 virus enhances the replicative fitness of Mx10 in the mouse model. Effect on oseltamivir treatment outcome in Mx10-infected mice by a shift in HA receptor specificity. The receptor binding region of the HA protein is critical to determining whether the virion will preferentially bind to ␣2,6-SA or ␣2,3-SA, the preferred receptors for human and avian viruses, respectively. It has been demonstrated that a D222G (H1 numbering) mutation in pH1N1 viruses is responsible for adaptation and more severe disease in mice and may be linked to more severe outcome in humans (37, 45–52). The pH1N1 viruses show altered receptor specificity, due to the D222G mutation, resulting in increased affinity for the ␣2,3-SA receptor. In agreement with previous reports (8, 9), when we mutated residue 222 to glycine (Mx10/HAD222G) we observed an increase in weight loss and disease signs in PBS-treated mice compared to wild-type Mx10-infected mice (Fig. 4). Interestingly, Mx10/HAD222G-infected mice treated with oseltamivir showed reduced weight loss and disease signs compared to PBStreated mice, indicating that oseltamivir had a therapeutic effect (Fig. 4), in contrast to the outcome with the wild-type Mx10 virus. These results show that a change in receptor specificity was sufficient to cause a change in treatment outcomes for Mx10 infections. Therefore, it appears that altering the functional balance between HA receptor attachment and NA sialidase activity is sufficient to alter the ability of oseltamivir to enhance the fitness of the virus for replication in mice, a finding indicative of the finely tuned balance between HA and NA activities needed to promote a pathogenic outcome. DISCUSSION

We have demonstrated that oseltamivir treatment of mice infected with pH1N1 viruses exacerbates the usual course of mild disease, transforming it into a highly pathogenic infection. This outcome was shown in detail using the pH1N1 virus isolate A/InDRE/Mexico/4487/2009 (Mx10) that has the wild-type sequence previously reported for the clinical isolate. This outcome was fur-

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FIG 3 Weight loss and lung virus titers of influenza virus-infected BALB/c

mice. (a) Mice were infected with Mx10 (n ⫽ 24), oseltamivir-resistant Mx10 (Mx10/NAH275Y) (n ⫽ 8), or an Mx10 recombinant virus containing USSR/ 90/77 NA (Mx10/U77NA) (n ⫽ 12). The percent body weights and standard deviations were calculated, and clinical signs were recorded for 17 days. (b) Mx10 (n ⫽ 12/group)-, Mx10-NAH275Y (n ⫽ 3/group)-, and Mx10/U77NA (n ⫽ 3/group)-infected animals were sacrificed at 3, 6, and 8 dpi, and the lung virus titers were calculated. The results are reported as means ⫾ the SEM. **, No surviving Mx10/U77NA-infected mice by 8 dpi.

ther confirmed with the wild-type A/California/04/2009 (Cal04) virus, the prototypic strain that has been studied extensively and well as additional clinical isolate, A/Canada-AB/RV1532/2009. Mice infected with any of the pH1N1 viruses and treated with oseltamivir showed development of severe disease and weight loss. Furthermore, when we modified the dose schedule or the amount of oseltamivir administered, the same phenomenon was observed. Even when the dose administered was changed from 50 to 2 mg/ kg/day, the mice developed a severe disease requiring euthanasia, indicating that this low dose of oseltamivir was likely already at a saturated level in the mice to confer this progression of disease. Our experiments with the Mx10 virus demonstrated elevated viral loads in the lungs with increased virus-induced lung pathology and antigen distribution in mice treated with oseltamivir compared to infected mice treated with PBS. In an effort to delineate the mechanism responsible for this phenomenon, we determined that the incorporation of mutations and resistance markers to

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FIG 4 Mx10/HAD222G-infected mice treated with oseltamivir. Mice were

infected with Mx10 (n ⫽ 24/treatment group) or Mx10/HAD222G (n ⫽ 6/treatment group) and were treated with either PBS or oseltamivir at 50 mg/kg/day for 5 days starting 1 day prior to infection. The percent body weights and standard deviations were calculated, and clinical signs were recorded for 17 days.

oseltamivir were not a factor in the increased virulence observed, and that these viruses were fully susceptible to oseltamivir pressure, as seen by the inhibition of in vitro viral growth and NA enzymatic activity. Our data have shown that the change in the course of illness in mice correlates with a shift in the HA-NA activity balance whether this occurs due to a reduction of NA enzymatic activity by oseltamivir treatment, the exchange of the wild-type NA for a lower-activity NA, or a mutation in HA that alters the receptor binding preference. It is well known that a balance between HA and NA activities is necessary for viral fitness. Furthermore, it has been shown that pH1N1 viruses have a lowactivity NA that is complemented by a HA with low affinity to sialic acid (53). However, this is the first time it has been reported that a further reduction of NA activity caused increased virulence of pH1N1 viruses in mice, apparently by enhancing the HA-NA activity balance in that animal species. The enzymatic activity of NA removes sialic acid (SA) from host cell glycoproteins and glycolipids and newly synthesized viral glycoproteins to facilitate virus budding and spread to neighboring cells. Efficient viral replication is dependent on the maintenance of a critical balance between the activities and specificities of HA and NA (54–56). Influenza virus HA mediates attachment of virions to cells by specific interaction with SA on cell surface glycoconjugates. Human strains of influenza preferentially bind SA with a ␣2,6-linkage to penultimate galactose, whereas avian strains of influenza prefer ␣2,3-SA (57, 58). pH1N1 viruses preferentially bind ␣2,6-SA but also exhibit efficient binding to ␣2,3-SA (59). Together, the activities of both the HA and the NA proteins contribute to host range, tissue tropism, and viral pathogenesis, and the balance between these activities can influence virulence (54). Typically, a drug that strongly inhibits NA activity, such as oseltamivir, will significantly disrupt the balance between HA and NA activities, and budding virions will remain attached to cellular glycoconjugates or remain aggregated by interaction of HA with sialic acid on the new synthesized viral glycoproteins (10, 11). However, in the treatment of pH1N1 infection in mice with

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oseltamivir, there was an apparent increase in the fitness of viral replication resulting in enhanced virulence, suggesting that a reduction in NA activity actually resulted in a better functional balance between HA and NA activities. To test this hypothesis, we generated two pH1N1 viruses that had lower NA activities than the wild-type pH1N1, Mx10/NAH275Y and Mx10/U77NA, while maintaining the HA. Introducing a single point mutation known to reduce NA activity, H275Y, into the Mx10 NA gene or replacing the entire NA with one that has significantly lower enzymatic activity was sufficient to increase virulence in mice. A similar correlation between decreased NA activity and increased infectivity in mice was previously reported, although increased pathogenicity was not reported with the pH1N1 strain used in that study (60). Another study supporting our findings demonstrated increased viral titers from mouse lungs infected with pH1N1 oseltamivirresistant viruses compared to their wild-type counterpart (61). Finally, a study comparing reduced NA activity by introducing the H275Y mutation into pH1N1 viruses showed accelerated weight loss, suggesting an increase in viral pathogenicity (62). However, to our knowledge, this is the first study using a mouse model to demonstrate that treatment with oseltamivir can cause an increase in viral fitness leading to a lethal outcome. A change in the affinity of HA for its receptor will also alter the dynamics between HA and NA functionality. Therefore, it is expected that the resulting shift in HA-NA functional balance could alter viral virulence and the therapeutic efficacy of oseltamivir (55). We tested this idea by treating mice with oseltamivir that had been infected with the Mx10/HAD222G variant. This mutation has been shown to be critical for pH1N1 mouse adaptation by shifting receptor specificity from ␣2,6-SA to ␣2,3-SA (8, 9, 37, 45–52) and results in increased virulence in the mouse model. Under these conditions, oseltamivir treatment showed a therapeutically beneficial effect compared to mock treatment. These results are in agreement with previous studies, which used a mouse-adapted Cal04 virus to study the efficacy of oseltamivir against a pH1N1 virus in mice (63). Similar to the Mx10/ HAD222G virus, the mouse-adapted Cal04 virus contains mutations in HA, including the D222G mutation, that increase its affinity for ␣2,3-SA (63), the primary species of SA that is expressed in the lower respiratory tracts of mice (64, 65), and reduces affinity for ␣2,6-SA (37, 45, 46, 66). This change in receptor specificity of HA would consequently alter the dependence of viral replication in mice on the level of NA enzymatic activity (35). Following a similar line of reasoning, it would be expected that treatment of pH1N1 infection in other animal species may result in a different outcome than that observed in mice as a consequence of speciesspecific differences in SA expression in their respiratory tissues. Unlike mice, ferrets predominantly express ␣2,6-SA in their respiratory tracts (64, 65), again shifting the balance between HA and NA activities required for optimal virus growth in that host compared to the growth requirements in mice. In agreement with this idea, previous studies have reported successful oseltamivir treatment of ferrets infected with wild-type Cal04, as evaluated by a reduction in weight loss and the severity of disease compared to mock-treated animals (16). Thus, the effects of NA activity reduction by drug treatment can be both virus and host dependent. However, these results also highlight possible pitfalls of using only a single animal model to assess the therapeutic efficacy of oseltamivir treatment, which has differences in its receptor type and distribution compared to humans. The effectiveness of oseltami-

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Neuraminidase Activity Alters Influenza Disease

vir against new and emerging strains of influenza is frequently tested in mice and other animal species and is used as a predictor for efficacy in humans (12–14, 67–80), but we demonstrate here that evaluation of antiviral efficacy in in vitro assays as well as in vivo analyses in other animal species should be assessed collectively to ensure that an accurate understanding of antiviral efficacy for humans will be predicted. The unexpected outcome attending oseltamivir treatment in pH1N1-infected mice presents an important extension that suggests there may be need for increased vigilance attending the use of oseltamivir, and potentially other NA inhibitors, since inadvertent changes in the interaction between the virus and host could confound the intended therapeutic benefit. Although, to date, this effect has never been reported in humans and remains a phenomenon only observed in mice, it is dependent on the interaction between the host SA receptor type and distribution and the fitness of the virus in a host as a function of its HA-NA activity balance. At this time, too little is understood about this interaction between virus and host to predict whether a virus will exhibit this phenomenon in a given host, although it is clear that infection with a virus that exhibits a well-adapted HA-NA activity for a host will most likely be amenable to NA inhibitor treatment since treatment will upset the optimal HA-NA balance. The phenomenon we have described may be particularly significant for zoonotic influenza viruses that have recently entered the human population and have HA and NA activities that are not yet highly adapted to receptors in the human respiratory tract. Increased vigilance may be a necessary precaution to ensure the usefulness of NA inhibitors for the treatment of novel zoonotic influenza viruses. ACKNOWLEDGMENTS

7.

8.

9.

10. 11. 12.

13.

14.

15.

We thank the Genomics Core at the National Microbiology Laboratory in Winnipeg for their assistance with primer synthesis and DNA sequencing.

FUNDING INFORMATION This research was funded by the Public Health Agency of Canada.

16.

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