Virus Research 185 (2014) 41–46
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Mammalian pathogenesis of oseltamivir-resistant pandemic (H1N1) 2009 influenza virus isolated in South Korea Donghyok Kwon 1 , Kyeongcheol Shin, Su-Jin Kim, Joo-Yeon Lee, Chun Kang ∗ Division of Influenza Virus, Center for Infectious Diseases, Korea National Institute of Health, Osong, Chungcheongbuk-do 363-951, South Korea
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Article history: Received 25 November 2013 Received in revised form 25 February 2014 Accepted 11 March 2014 Available online 19 March 2014 Keywords: Pandemic (H1N1) 2009 Influenza Oseltamivir-resistant Ferrets Mice Pathogenicity
a b s t r a c t Oseltamivir, a neuraminidase (NA) inhibitor, has been widely used for the treatment of patients infected with the pandemic (H1N1) 2009 influenza virus. With the increasing use of oseltamivir, drug-resistant mutants emerged rapidly and 11 cases of resistant viruses were detected during the 2009 H1N1 pandemic in South Korea. To better understand the differences between oseltamivir-susceptible and oseltamivir-resistant virus, we compared the replication and pathogenesis of the NA H275Y mutant virus, A/Gyeongnam/1820/2009, in ferrets and mice with those of oseltamivir-susceptible A/Korea/01/2009 virus. Oseltamivir-resistant virus infected ferrets showed mild clinical signs and the virus replicated well in the upper respiratory tract and slightly in the lower respiratory tract. No virus was detected in the extrapulmonary organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected mainly in the bronchiolar epithelial cells, cells present in the interstitial septa, pneumocytes and peribronchial glands with severe peribronchitis. A/Gyeongnam/1820/2009 virus-infected mice showed weight loss and the virus replicated in lungs with high titer. Histopathologically, the mice showed mild to moderate alveolitis, interstitial pneumonia and perivascular lymphoid tissue hyperplasia. In lungs, bronchiolar epithelial cells, pneumocytes and interstitial inflammatory cells were infected by influenza virus and trachea epithelial cells were the sites of infection. When compared with the results of A/Korea/01/2009 oseltamivir-susceptible pandemic influenza virus, an oseltamivir-resistant virus isolated in South Korea showed fewer pathogenic properties in ferrets and similar fitness in mice. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Since the first report of pandemic influenza A(H1N1) 2009 (H1N1pdm09) virus in April 2009, the virus has spread worldwide and caused a pandemic in the 21st century (Dawood et al., 2009). Anti-influenza agents have been widely used for the
Abbreviations: NA, neuraminidase; HA, hemagglutinin; M, matrix; NAIs, neuraminidase inhibitors; H1N1pdm09, pandemic influenza A(H1N1) 2009; BSL3, biosafety level 3; KCDC, Korea Centers for Disease Control and Prevention; A/GN/1820, A/Gyeongnam/1820/2009; A/KR/01, A/Korea/01/2009; MDCK, Madin–Darby canine kidney; PFU, plaque forming unit; MUNANA, 2 (4-methylumbelliferyl)-␣-d-N-acetylneuraminic acid; PBS, phosphate-buffered saline; HI, hemagglutination inhibition; p.i., post inoculation; RII, relative inactivity index; MID, mouse infectious dose; MLD, mouse lethal dose; H&E, hematoxylin and eosin. ∗ Corresponding author. Tel.: +82 43 719 8190; fax: +82 43 719 8219. E-mail addresses: [email protected], [email protected] (C. Kang). 1 Present address: Division of Epidemic Intelligence Service, Korea Centers for Disease Control and Prevention, Osong, Chungcheonbuk-do 363-951, South Korea. http://dx.doi.org/10.1016/j.virusres.2014.03.014 0168-1702/© 2014 Elsevier B.V. All rights reserved.
chemoprophylaxis and treatment during the 2009 H1N1 pandemic. Due to natural resistance of H1N1pdm09 viruses against adamantanes, neuraminidase (NA) inhibitors (NAIs), especially oseltamivir, were broadly used to mitigate morbidity and mortality during the early phase of the pandemic. With the increasing use of antivirals, oseltamivir-resistant mutants emerged rapidly and 11 cases of resistant viruses were detected during the H1N1 pandemic in South Korea (Shin et al., 2011; Yi et al., 2010). The oseltamivir-resistant viral infections were observed mainly in immunocompromised patients treated with antivirals and all the NAI-resistant variants to H1N1pdm09 viruses had H275Y (N1 numbering) substitution at NA gene (Yi et al., 2010). To assess the fitness and transmissibility of the newly identified H1N1pdm09 virus, various mammalian models have been used and the results were similar or different depending on the viruses used (Itoh et al., 2009; Kwon et al., 2010; Lange et al., 2009; Maines et al., 2009; Munster et al., 2009). The transmissibility and pathogenicity of several strains of oseltamivir-resistant H1N1pdm09 virus also have been studied in vitro and/or in vivo and showed inconsistent results according to the isolates tested (Duan et al., 2010; Hamelin
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et al., 2010; Kiso et al., 2010; Memoli et al., 2011; Seibert et al., 2010). To characterize the replication and pathogenic properties of the Korean oseltamivir-resistant H1N1pdm09 virus, we performed animal study using mammalian model, ferrets and mice. 2. Materials and methods 2.1. Ethics statement All experimental animal works were performed in the biosafety level 3 (BSL3) animal facilities at Korea Centers for Disease Control and Prevention (KCDC), followed with approval of the animal safety protocols by the Institutional Animal Care and Use Committee of KCDC. 2.2. Viruses A/Gyeongnam/1820/2009 (A/GN/1820) virus was isolated and was propagated in the Madin–Darby canine kidney (MDCK) cells using nasal and oropharyngeal swabs from 1 year old female patient who died in spite of oseltamivir and peramivir treatment after hospitalization. She had myelodysplasia as an underlying disease. The virus had a titer of 107.1 plaque forming units (PFU)/ml. The amino acid substitution of histidine at position 275 in the NA into tyrosine of this virus was confirmed by sequence analysis and the dose of oseltamivir and zanamivir required for 50% inhibition (IC50 ) of NA activity was measured by phenotypic assay using a fluorescent substrate 2 -(4methylumbelliferyl)-␣-d-N-acetylneuraminic acid (MUNANA) (Yi et al., 2010). Oseltamivir-susceptible A/Korea/01/2009 (A/KR/01) virus which was isolated from index case of South Korea was used as a control. A/KR/01 was propagated in the MDCK cells and had a titer of 107.4 PFU/ml. All experiments with these viruses were performed at a BSL3 facility in KCDC. The genome sequences of hemagglutinin (HA), NA and matrix (M) gene of A/GN/1820 virus was submitted to GenBank with accession numbers CY060466–CY060468. Oseltamivir carboxylate was provided by the F. Hoffmann-La Roche Ltd. 2.3. Virus infection in ferrets To evaluate the pathogenicity of oseltamivir-resistant and susceptible H1N1pdm09 virus in vivo, we used 6- to 8-month-old male ferrets (Triple F Farms, Sayre, PA, USA), which were serologically negative for currently circulating human influenza A(H1N1), A(H3N2), A(H1N1)pdm09 viruses, and influenza B viruses by hemagglutination inhibition (HI) assays. Eight animals were allocated randomly for testing each virus and baseline body weights and body temperature were measured before inoculation. Body temperatures were measured using a subcutaneous implantable temperature transponder (Bio Medic Data Systems Inc., Seaford, DE, USA). Ferrets were anesthetized through intramuscular injection of a cocktail of ketamine, xylazine and atropine. Six animals were inoculated intranasally with 107 PFU of oseltamivir-resistant mutant, A/GN/1820, or oseltamivir-sensitive wild-type virus, A/KR/01, in 1 ml sterile phosphate-buffered saline (PBS). Two control animals were mock-inoculated with sterile PBS.
at −80 ◦ C and for histological study, those were fixed in 10% neutralbuffered formalin until processing. Virus replication in the lungs, nasal turbinates and major organs were measured by plaque assays in MDCK cells. The remaining animals were monitored daily for clinical signs, weights and temperature for 14 days. A scoring system of 0–3 was used to assess the activity of inoculated ferrets and a relative inactivity index (RII) was calculated as described previously (Reuman et al., 1989; Zitzow et al., 2002). Nasal washes were collected every other day for 9 days after inoculation and the virus titer was determined in MDCK cells. 2.5. Pathogenicity in mice Female 6- to 7-week-old BALB/c mice (Orient Bio, South Korea) were anesthetized with isoflurane and inoculated intranasally with 50 l of A/GN/1820 or A/KR/01 virus. The 50% mouse infectious dose (MID50 ) and 50% mouse lethal dose (MLD50 ) were calculated by inoculating eight mice per group with serial 10-fold dilutions, from 10◦ to 106 PFU, of virus. After 3 days, lungs of three mice from each group were collected after euthanasia and the viral titers were measured in MDCK cells for the calculation of MID50 by the method of Reed and Muench (Reed and Muench, 1938). The remaining five mice were monitored daily for body weight change and mortality for 14 days. To determine the pathogenicity and replication fitness of the virus, six mice were inoculated with 105 PFU of each virus and three were euthanized on days 3 and 6 p.i. Systemic organs were collected aseptically and frozen on dry ice immediately for virus titration and fixed in 10% neutral-buffered formalin for histological analysis. 2.6. Histological and immunohistochemical investigation Histopathological analyses of animal tissues were performed as described previously (Kwon et al., 2010, 2011). In short, after fixation in 10% neutral-buffered formalin, tissues were routinely processed and embedded in paraffin. The paraffin blocks were cut into 5 m-thick sections and stained with hematoxylin and eosin (H&E) for histopathological analysis. For the evaluation of influenza viral antigen expression, tissues were processed for immunohistochemical staining with influenza A specific antibodies. A monoclonal antibody (Novus Biologicals, Littleton, CO, USA) against the nucleoprotein of influenza A virus and goat polyclonal anti-influenza A antibody (Millipore, Billerica, MA, USA) were applied for the tissues of ferret and mouse, respectively. Then the sections were treated with biotinylated anti-mouse and anti-goat IgGs (Dako, Glostrup, Denmark), respectively. The slides were then applied with alkaline phosphatase conjugated streptavidin (Merck KGaA, Darmstadt, Germany) and were visualized by a solution of red substrate (Roche Diagnostics GmbH, Mannheim, Germany). The sections were lightly counterstained with Mayer’s hematoxylin and mounted with aqueous mount (Dako, Glostrup, Denmark). 2.7. Hemagglutination Inhibition assays Pre-exposure sera were collected before virus inoculation and post-exposure sera were collected at day 21 p.i. from ferrets and at day 14 p.i. from mice and H1-specific antibodies were evaluated by HI assays using 0.5% turkey red blood cells as described previously (Kwon et al., 2010).
2.4. Monitoring and collection of ferret specimens 3. Results Three infected ferrets and one mock-infected ferret were euthanized at day 3 post inoculation (p.i.) and tissue specimens (lung, trachea, nose, spleen, kidney, liver, heart, intestine, and brain) were collected for viral titration and histopathological analysis. For viral titration, specimens were immediately frozen on dry ice and stored
3.1. Sequence analysis of two viruses To evaluate the similarity of two viruses at molecular level, gene sequences of 8 fragments of each virus were analyzed. When
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Table 1 Phenotypic and genotypic characteristics of oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus. NA enzyme inhibition IC50 a (nM)
Virus
A/Korea/01/2009 A/Gyeongnam/1820/2009 a
Amino acid at NA 275
Oseltamivir carboxylate
Zanamivir
1.18 359.4
0.42 0.78
His Tyr
IC50 value: 50% inhibitory concentration (nM) of duplicate assays.
Table 2 Clinical signs of oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus infected ferrets. Virus
Sneezing (no/total)
Nasal discharge (no/total)
Lethargy (RII)
Weight loss (%)
Temperature rise (◦ C)
A/Korea/01/2009 A/Gyeongnam/1820/2009
3/3 1/3
2/3 2/3
1.8 1.5
5.8 3.9
1.2 1.8
compared with the sequences of A/KR/01 virus, A/GN/1820 virus had a total of 11 amino acid changes in the PB2 (L154I), PB1 (V645I), PA (K142M, K298E and S632A), HA (S220T), NA (N248D, H275Y, R319S and I467V) and NS (I123V) genes. As far as we know these changes were not known to be related with virulence or antiviral drug resistance except for H275Y in the NA gene. 3.2. In vitro sensitivity of viruses to NA inhibitors For the assessment of the in vitro sensitivity to NA inhibitors of A/GN/1820 and A/KR/01 virus, we measured NA activity using oseltamivir carboxylate and zanamivir and determined IC50 values. The IC50 value of A/GN/1820 virus against oseltamivir was about 300-fold higher than that of A/KR/01, however the sensitivity to zanamivir was comparable (Table 1). 3.3. Pathogenicity of viruses in ferrets Ferrets inoculated with oseltamivir-resistant, A/GN/1820, or oseltamivir-susceptible, A/KR/01, virus showed mild lethargy, infrequent sneezing and nasal discharge. All ferrets lost minimal weight, and body temperature rose at 1 or 2 day p.i. (Table 2). At day 3 p.i., A/GN/1820 virus replicated to high titer in the upper respiratory tract (nasal turbinates), while only two of three ferrets showed low levels of virus replication in the lower respiratory tract (lungs). No virus was identified from extrapulmonary organs of ferrets by plaque assays. In contrast, A/KR/01 virus replicated to high titer in the nasal turbinates and lungs, and to low titer in brain of ferrets (Table 3). In the nasal wash, both viruses replicated efficiently for 5 days with some differences in virus replication pattern on 5 day p.i. (Fig. 1). Serum HA antibodies against both viruses were elevated to 1:640 to 1:1280 at day 21 p.i. by HI assays. Histopathologically, two viruses caused similar microscopic lesions in the lungs of ferrets at day 3 p.i. Mild to moderate bronchopneumonia and severe interstitial pneumonia by thickening of the alveolar septa with extensive inflammatory cell infiltrates were detected and perivascular lymphoid tissue hyperplasia around the
Fig. 1. Viral titers of nasal washes from ferrets inoculated with oseltamivirsusceptible or oseltamivir-resistant pandemic (H1N1) 2009 influenza virus. Virus titers were determined every other day over 9 days post inoculation (p.i.) from nasal washes of ferrets inoculated with each virus. Error bars indicate the standard deviations of viral titers of three ferrets. Detection limit: 1.5 log10 PFU/ml.
blood vessels was found (Fig. 2A and B). We assessed influenza A viral antigen expression by the method of immunohistochemistry and both viruses showed similar pattern. Bronchial and bronchiolar epithelial cells, inflammatory cells in the bronchiolar lumen were stained clearly (Fig. 2C and D). We could detect viral antigen frequently in cells located in the thickened alveolar wall and pneumocytes. Peribronchial glands with severe peribronchitis were also infected by both viruses (Fig. 2E and F). Influenza A viral antigen was not detected in any other extrapulmonary tissues in ferrets inoculated with oseltamivir-resistant or oseltamivir-susceptible pandemic H1N1 influenza virus. 3.4. Pathogenicity of viruses in mice To evaluate pathological changes in mice we inoculated mice with serial 10-fold dilutions of each influenza virus. 104 PFU or more amounts of A/GN/1820 virus-infected mice showed weight
Table 3 Virus titers in organs of ferrets inoculated with oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus. Virus
Virus titer (mean log10 PFU ± SD/g)a Respiratory tract
A/Korea/01/2009 A/Gyeongnam/1820/2009
Brain
Nasal turbinates
Lung
7.5 ± 0.2 6.6 ± 0.1
6.7 ± 0.4 3.7/4.0/–
Trachea b
ND 3.0 ± 0.9
Olfactory bulb c
2.0/3.6/– –/–/–
Anterior part 3.9/–/– –/–/–
a Three ferrets were euthanized at day 3 post infection for viral titration. When viruses were recovered from all three ferrets, mean titers are presented. When viruses were not recovered from all animals, individual titers are shown. b ND: not done. c Virus not detected (detection limit: 1.5 log10 PFU/g of tissue).
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Fig. 2. Histopathologic changes and viral antigen expression in the lungs of inoculated with oseltamivir-susceptible or oseltamivir-resistant pandemic (H1N1) 2009 influenza virus. Ferrets were inoculated with 107 PFU of A/KR/01 (A, C, and E) or A/GN/1820 (B, D, and F) intranasally and were euthanized on day 3 p.i. and the tissues were processed for Hematoxylin and Eosin (A and B) and immunohistochemical staining (C–F). (A and B) Prominent alveolar thickening with extensive inflammatory cell infiltrates and suppurative exudates in the bronchiolar lumen. Magnification, 50×. (C and D) Extensive viral antigens (red stain) were detected in the bronchial epithelial cells (arrows). Magnification, 100×. (E and F) Viral antigens were detected in the peribronchial glands (arrowheads). Magnification, 200×.
loss by 7–8 day p.i. and the MID50 was 101.75 PFU. However, all mice survived during the period of the experiment at the highest dose tested (MLD50 > 106 PFU). 105 PFU or more amounts of A/KR/01 virus-infected mice also showed weight loss by 7 day p.i. and the MID50 was 101.5 PFU and all mice survived like A/GN/1820 virus. At day 3 and 6 p.i., both viruses replicated to high titer in the lungs of mice. A/GN/1820 virus replicated in trachea moderately and one of three mice had low titer of virus in the nose at 3 day p.i. (Table 4). No virus was detected in extrapulmonary organs. A/GN/1820 or A/KR/01 virus-infected mouse lung tissues showed mild to moderate multifocal necrotizing bronchiolitis and interstitial pneumonia. Prominent alveolar thickening with extensive inflammatory infiltrates was detected and perivascular cuffing with accumulation of lymphocytes around the blood vessels was found at day 3 p.i. (Fig. 3B and C). Positive staining by immunohistochemistry in tissues of mice was confined to lung and trachea. Viral antigen was detected extensively in bronchiolar epithelial cells,
pneumocytes and cells in the thickened alveolar wall (Fig. 3D and E). We could find several positive viral antigen signals in the tracheal epithelial cells (Fig. 3F), but not in any other extrapulmonary tissues. 4. Discussion During the 2009 H1N1 pandemic anti-influenza drugs were used extensively for the treatment and prophylaxis of influenza virus infection in South Korea. Korea CDC identified 11 cases of oseltamivir-resistant influenza virus among patients who had influenza that was refractory to antiviral treatment in South Korea (Shin et al., 2011; Yi et al., 2010). Because three of the 11 cases were fatal it was needed to assess the characteristics of the antiviralresistant H1N1pdm09 virus to compare the pathogenicity with antiviral-sensitive counterpart in animal models. In this study, we investigated the mammalian pathogenicity of oseltamivir-resistant H1N1pdm09 virus, A/GN/1820, which was
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Table 4 Virus titers in respiratory organs of mice inoculated with oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus. Virus
Virus titer (mean log10 PFU ± SD/g)a Lung
A/Korea/01/2009 A/Gyeongnam/1820/2009
Trachea
Nose
Day 3
Day 6
Day 3
Day 6
Day 3
Day 6
6.4 ± 0.4 7.2 ± 0.1
5.7 ± 0.3 6.7 ± 0.4
NDb 5.2 ± 0.2
ND 4.4 ± 0.1
2.9/2.7/–c 3.8/–/–
–/–/– –/–/–
a Three mice were euthanized at day 3 and 6 post infection for viral titration. When viruses were recovered from all three mice, mean titers are presented. When viruses were not recovered from all animals, individual titers are shown. b ND: not done. c Virus not detected (detection limit: 1.5 log10 PFU/g of tissue).
isolated from lethal case during 2009 H1N1 pandemic in South Korea. When compared with A/KR/01 virus which was the first isolate in South Korea in 2009, the amino acid sequences of A GN/1820 virus were changed at 11 sites in the PB2, PB1, PA, HA, NA and NS genes. However, we couldn’t find the changes related with increasing the virulence or antiviral drug resistance except for the H275Y mutation in the NA gene. Genotypic and phenotypic assay showed that this virus had H275Y mutation in NA gene and the IC50 value was about 300 times higher than that of sensitive counterpart A/KR/01 however A/GN/1820 virus showed sensitivity to zanamivir. To evaluate the fitness of oseltamivir-resistant mutant virus which caused mortality in human, we performed in vivo experiment using ferrets and mice and compared the results with those of antiviral-susceptible wild-type virus. Our study revealed that oseltamivir-resistant H1N1pdm09 virus isolated in South Korea replicated to similar levels with wildtype virus in upper respiratory tract however this mutant virus showed less efficient replication pattern in the lower respiratory tract of ferrets. Among three ferrets inoculated with A/GN/1820 virus, only two showed low level of virus replication in the lungs whereas all three A/KR/01 virus-inoculated ferrets showed high level of virus replication in lungs. Although both viruses caused similar clinical changes and histopathological pulmonary lesions in ferrets, oseltamivir-resistant mutant virus showed reduced
viral replication in ferret lungs. In mouse model oseltamivirresistant virus showed no significant difference in pathogenicity when compared with oseltamivir-susceptible virus however it required 10 times much amount of virus to cause weight loss. Immunohistochemical analysis provides valuable information on the pathogenesis of influenza virus. We could identify the replication sites and cell types in tissues of influenza virus infected animals. Although A/GN/1820 virus replicated less efficiently than A/KR/01 virus in ferrets lungs, the target cells were very similar. Both viruses replicated in bronchial/bronchiolar epithelial cells, interstitial macrophages, pneumocytes and peribronchial glands of ferrets. In mice, these viruses replicated mainly in bronchiolar epithelial cells, pneumocytes, interstitial inflammatory cells and tracheal epithelial cells. Chronological analysis showed that A/GN/1820 virus replicated highly in the mouse lungs from day 1 to day 7 p.i. without previous adaptation (data not shown). This result indicated that the pandemic H1N1 viruses whichever oseltamivir-resistant or oseltamivir-susceptible, could infect mice more efficiently than seasonal influenza viruses. Although this mutant virus was isolated from lethal infant case, the pathogenic properties in mammalian models showed less reduced viral fitness when compared with wild-type virus. The viral fitness, pathogenicity and transmission of several strains of the oseltamivir-resistant pandemic (H1N1) 2009 influenza virus have been studied in vitro and/or in vivo and the results have
Fig. 3. Representative histopathology and immunohistochemical results in respiratory organs from mice inoculated with oseltamivir-susceptible or oseltamivir-resistant pandemic (H1N1) 2009 influenza virus. Tissues were removed on 3 or 6 day p.i. and were processed for H&E (A–C) and immunohistochemical staining (D–F). (A) Mockinfected mouse lung showing normal histology. Magnification, 100×. (B and C) Lungs of A/GN/1820 virus-infected mice show prominent alveolar thickening with extensive inflammatory infiltrates (B) and perivascular cuffing (C) at day 6 p.i. Magnification, 100×. (D and E) Immunohistochemical staining of A/KR/01 (D) and A/GN/1820 (E) virus infected mouse lung at day 3 p.i. Influenza antigens were detected in the bronchiolar epithelial cells (arrows) and pneumocytes (arrowheads). Magnification, 200×. (F) Viral antigen was detected in A/GN/1820 virus-infected mouse tracheal epithelial cells (arrows). Magnification, 200×.
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shown variable pathogenic properties depending on the isolates used when compared with the oseltamivir-sensitive counterparts (Duan et al., 2010; Hamelin et al., 2010; Kiso et al., 2010; Memoli et al., 2011; Seibert et al., 2010). Previous studies revealed that the oseltamivir-resistant viruses possess lower or equivalent transmissibility in ferrets depending on the isolates (Duan et al., 2010; Kiso et al., 2010; Seibert et al., 2010). However we do not know if the Korean mutant virus can be transmissible in ferrets because we could not perform transmission experiments and this is the limitation in our study and needs further study. The extensive usage of oseltamivir may trigger the emergence of NA inhibitor-resistant mutants which possess efficient replication fitness, virulence and transmissibility in humans. In South Korea, most of the oseltamivir-resistant viruses were identified in patients with underlying diseases and caused death in 30 per cent (Yi et al., 2010). Therefore it is very important to find antiviralresistant mutant viruses through national surveillance system and it needs more caution when treat patients with underlying disease. Conflict of interest There are no actual or potential conflicts of interest. Acknowledgements This study was supported by the Intramural Research Program (2010-N43001-00) of the Korea National Institute of Health, Korea Centers for Disease Control and Prevention. References Dawood, F.S., Jain, S., Finelli, L., Shaw, M.W., Lindstrom, S., Garten, R.J., Gubareva, L.V., Xu, X., Bridges, C.B., Uyeki, T.M., 2009. Emergence of a novel swine-origin influenza a (H1N1) virus in humans. N. Engl. J. Med. 360, 2605–2615. Duan, S., Boltz, D.A., Seiler, P., Li, J., Bragstad, K., Nielsen, L.P., Webby, R.J., Webster, R.G., Govorkova, E.A., 2010. Oseltamivir-resistant pandemic H1N1/2009 influenza virus possesses lower transmissibility and fitness in ferrets. PLoS Pathog. 6, e1001022. Hamelin, M.E., Baz, M., Abed, Y., Couture, C., Joubert, P., Beaulieu, E., Bellerose, N., Plante, M., Mallett, C., Schumer, G., Kobinger, G.P., Boivin, G., 2010. Oseltamivirresistant pandemic A/H1N1 virus is as virulent as its wild-type counterpart in mice and ferrets. PLoS Pathog. 6, e1001015.
Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y., Tamura, D., Sakai-Tagawa, Y., Noda, T., Sakabe, S., Imai, M., Hatta, Y., Watanabe, S., Li, C., Yamada, S., Fujii, K., Murakami, S., Imai, H., Kakugawa, S., Ito, M., Takano, R., Iwatsuki-Horimoto, K., Shimojima, M., Horimoto, T., Goto, H., Takahashi, K., Makino, A., Ishigaki, H., Nakayama, M., Okamatsu, M., Warshauer, D., Shult, P.A., Saito, R., Suzuki, H., Furuta, Y., Yamashita, M., Mitamura, K., Nakano, K., Nakamura, M., Brockman-Schneider, R., Mitamura, H., Yamazaki, M., Sugaya, N., Suresh, M., Ozawa, M., Neumann, G., Gern, J., Kida, H., Ogasawara, K., Kawaoka, Y., 2009. In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature 460, 1021–1025. Kiso, M., Shinya, K., Shimojima, M., Takano, R., Takahashi, K., Katsura, H., Kakugawa, S., Le, M.T., Yamashita, M., Furuta, Y., Ozawa, M., Kawaoka, Y., 2010. Characterization of oseltamivir-resistant 2009 H1N1 pandemic influenza A viruses. PLoS Pathog. 6, e1001079. Kwon, D., Shin, K., Kim, S., Ha, Y., Choi, J.H., Yang, J.S., Lee, J.Y., Chae, C., Oh, H.B., Kang, C., 2010. Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models. J. Microbiol. 48, 657–662. Kwon, D., Shin, K., Shin, J.Y., Lee, J.Y., Ha, Y., Lee, N.J., Oh, H.B., Chae, C., Kang, C., 2011. Pathogenesis and chronologic localization of the human influenza A (H1N1) virus in cotton rats. Public Health Res. Perspect. 2, 15–22. Lange, E., Kalthoff, D., Blohm, U., Teifke, J.P., Breithaupt, A., Maresch, C., Starick, E., Fereidouni, S., Hoffmann, B., Mettenleiter, T.C., Beer, M., Vahlenkamp, T.W., 2009. Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1 after experimental infection of pigs. J. Gen. Virol. 90, 2119–2123. Maines, T.R., Jayaraman, A., Belser, J.A., Wadford, D.A., Pappas, C., Zeng, H., Gustin, K.M., Pearce, M.B., Viswanathan, K., Shriver, Z.H., Raman, R., Cox, N.J., Sasisekharan, R., Katz, J.M., Tumpey, T.M., 2009. Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science 325, 484–487. Memoli, M.J., Davis, A.S., Proudfoot, K., Chertow, D.S., Hrabal, R.J., Bristol, T., Taubenberger, J.K., 2011. Multidrug-resistant 2009 pandemic influenza A(H1N1) viruses maintain fitness and transmissibility in ferrets. J. Infect. Dis. 203, 348–357. Munster, V.J., de Wit, E., van den Brand, J.M., Herfst, S., Schrauwen, E.J., Bestebroer, T.M., van de Vijver, D., Boucher, C.A., Koopmans, M., Rimmelzwaan, G.F., Kuiken, T., Osterhaus, A.D., Fouchier, R.A., 2009. Pathogenesis and transmission of swineorigin 2009 A(H1N1) influenza virus in ferrets. Science 325, 481–483. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 27, 493–497. Reuman, P.D., Keely, S., Schiff, G.M., 1989. Assessment of signs of influenza illness in the ferret model. J. Virol. Methods 24, 27–34. Seibert, C.W., Kaminski, M., Philipp, J., Rubbenstroth, D., Albrecht, R.A., Schwalm, F., Stertz, S., Medina, R.A., Kochs, G., Garcia-Sastre, A., Staeheli, P., Palese, P., 2010. Oseltamivir-resistant variants of the 2009 pandemic H1N1 influenza A virus are not attenuated in the guinea pig and ferret transmission models. J. Virol. 84, 11219–11226. Shin, S.Y., Kang, C., Gwack, J., Kim, J.H., Kim, H.S., Kang, Y.A., Lee, H.G., Kim, J.S., Lee, J.K., Kim, S.H., 2011. Drug-resistant pandemic (H1N1) 2009, South Korea. Emerg. Infect. Dis. 17, 702–704. Yi, H., Lee, J.Y., Hong, E.H., Kim, M.S., Kwon, D., Choi, J.H., Choi, W.Y., Kim, K.S., Lee, J.K., Oh, H.B., Kang, C., 2010. Oseltamivir-resistant pandemic (H1N1) 2009 virus, South Korea. Emerg. Infect. Dis. 16, 1938–1942. Zitzow, L.A., Rowe, T., Morken, T., Shieh, W.J., Zaki, S., Katz, J.M., 2002. Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J. Virol. 76, 4420–4429.
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Mammalian pathogenesis of oseltamivir-resistant pandemic (H1N1) 2009 influenza virus isolated in South Korea Donghyok Kwon 1 , Kyeongcheol Shin, Su-Jin Kim, Joo-Yeon Lee, Chun Kang ∗ Division of Influenza Virus, Center for Infectious Diseases, Korea National Institute of Health, Osong, Chungcheongbuk-do 363-951, South Korea
a r t i c l e
i n f o
Article history: Received 25 November 2013 Received in revised form 25 February 2014 Accepted 11 March 2014 Available online 19 March 2014 Keywords: Pandemic (H1N1) 2009 Influenza Oseltamivir-resistant Ferrets Mice Pathogenicity
a b s t r a c t Oseltamivir, a neuraminidase (NA) inhibitor, has been widely used for the treatment of patients infected with the pandemic (H1N1) 2009 influenza virus. With the increasing use of oseltamivir, drug-resistant mutants emerged rapidly and 11 cases of resistant viruses were detected during the 2009 H1N1 pandemic in South Korea. To better understand the differences between oseltamivir-susceptible and oseltamivir-resistant virus, we compared the replication and pathogenesis of the NA H275Y mutant virus, A/Gyeongnam/1820/2009, in ferrets and mice with those of oseltamivir-susceptible A/Korea/01/2009 virus. Oseltamivir-resistant virus infected ferrets showed mild clinical signs and the virus replicated well in the upper respiratory tract and slightly in the lower respiratory tract. No virus was detected in the extrapulmonary organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected mainly in the bronchiolar epithelial cells, cells present in the interstitial septa, pneumocytes and peribronchial glands with severe peribronchitis. A/Gyeongnam/1820/2009 virus-infected mice showed weight loss and the virus replicated in lungs with high titer. Histopathologically, the mice showed mild to moderate alveolitis, interstitial pneumonia and perivascular lymphoid tissue hyperplasia. In lungs, bronchiolar epithelial cells, pneumocytes and interstitial inflammatory cells were infected by influenza virus and trachea epithelial cells were the sites of infection. When compared with the results of A/Korea/01/2009 oseltamivir-susceptible pandemic influenza virus, an oseltamivir-resistant virus isolated in South Korea showed fewer pathogenic properties in ferrets and similar fitness in mice. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Since the first report of pandemic influenza A(H1N1) 2009 (H1N1pdm09) virus in April 2009, the virus has spread worldwide and caused a pandemic in the 21st century (Dawood et al., 2009). Anti-influenza agents have been widely used for the
Abbreviations: NA, neuraminidase; HA, hemagglutinin; M, matrix; NAIs, neuraminidase inhibitors; H1N1pdm09, pandemic influenza A(H1N1) 2009; BSL3, biosafety level 3; KCDC, Korea Centers for Disease Control and Prevention; A/GN/1820, A/Gyeongnam/1820/2009; A/KR/01, A/Korea/01/2009; MDCK, Madin–Darby canine kidney; PFU, plaque forming unit; MUNANA, 2 (4-methylumbelliferyl)-␣-d-N-acetylneuraminic acid; PBS, phosphate-buffered saline; HI, hemagglutination inhibition; p.i., post inoculation; RII, relative inactivity index; MID, mouse infectious dose; MLD, mouse lethal dose; H&E, hematoxylin and eosin. ∗ Corresponding author. Tel.: +82 43 719 8190; fax: +82 43 719 8219. E-mail addresses: [email protected], [email protected] (C. Kang). 1 Present address: Division of Epidemic Intelligence Service, Korea Centers for Disease Control and Prevention, Osong, Chungcheonbuk-do 363-951, South Korea. http://dx.doi.org/10.1016/j.virusres.2014.03.014 0168-1702/© 2014 Elsevier B.V. All rights reserved.
chemoprophylaxis and treatment during the 2009 H1N1 pandemic. Due to natural resistance of H1N1pdm09 viruses against adamantanes, neuraminidase (NA) inhibitors (NAIs), especially oseltamivir, were broadly used to mitigate morbidity and mortality during the early phase of the pandemic. With the increasing use of antivirals, oseltamivir-resistant mutants emerged rapidly and 11 cases of resistant viruses were detected during the H1N1 pandemic in South Korea (Shin et al., 2011; Yi et al., 2010). The oseltamivir-resistant viral infections were observed mainly in immunocompromised patients treated with antivirals and all the NAI-resistant variants to H1N1pdm09 viruses had H275Y (N1 numbering) substitution at NA gene (Yi et al., 2010). To assess the fitness and transmissibility of the newly identified H1N1pdm09 virus, various mammalian models have been used and the results were similar or different depending on the viruses used (Itoh et al., 2009; Kwon et al., 2010; Lange et al., 2009; Maines et al., 2009; Munster et al., 2009). The transmissibility and pathogenicity of several strains of oseltamivir-resistant H1N1pdm09 virus also have been studied in vitro and/or in vivo and showed inconsistent results according to the isolates tested (Duan et al., 2010; Hamelin
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et al., 2010; Kiso et al., 2010; Memoli et al., 2011; Seibert et al., 2010). To characterize the replication and pathogenic properties of the Korean oseltamivir-resistant H1N1pdm09 virus, we performed animal study using mammalian model, ferrets and mice. 2. Materials and methods 2.1. Ethics statement All experimental animal works were performed in the biosafety level 3 (BSL3) animal facilities at Korea Centers for Disease Control and Prevention (KCDC), followed with approval of the animal safety protocols by the Institutional Animal Care and Use Committee of KCDC. 2.2. Viruses A/Gyeongnam/1820/2009 (A/GN/1820) virus was isolated and was propagated in the Madin–Darby canine kidney (MDCK) cells using nasal and oropharyngeal swabs from 1 year old female patient who died in spite of oseltamivir and peramivir treatment after hospitalization. She had myelodysplasia as an underlying disease. The virus had a titer of 107.1 plaque forming units (PFU)/ml. The amino acid substitution of histidine at position 275 in the NA into tyrosine of this virus was confirmed by sequence analysis and the dose of oseltamivir and zanamivir required for 50% inhibition (IC50 ) of NA activity was measured by phenotypic assay using a fluorescent substrate 2 -(4methylumbelliferyl)-␣-d-N-acetylneuraminic acid (MUNANA) (Yi et al., 2010). Oseltamivir-susceptible A/Korea/01/2009 (A/KR/01) virus which was isolated from index case of South Korea was used as a control. A/KR/01 was propagated in the MDCK cells and had a titer of 107.4 PFU/ml. All experiments with these viruses were performed at a BSL3 facility in KCDC. The genome sequences of hemagglutinin (HA), NA and matrix (M) gene of A/GN/1820 virus was submitted to GenBank with accession numbers CY060466–CY060468. Oseltamivir carboxylate was provided by the F. Hoffmann-La Roche Ltd. 2.3. Virus infection in ferrets To evaluate the pathogenicity of oseltamivir-resistant and susceptible H1N1pdm09 virus in vivo, we used 6- to 8-month-old male ferrets (Triple F Farms, Sayre, PA, USA), which were serologically negative for currently circulating human influenza A(H1N1), A(H3N2), A(H1N1)pdm09 viruses, and influenza B viruses by hemagglutination inhibition (HI) assays. Eight animals were allocated randomly for testing each virus and baseline body weights and body temperature were measured before inoculation. Body temperatures were measured using a subcutaneous implantable temperature transponder (Bio Medic Data Systems Inc., Seaford, DE, USA). Ferrets were anesthetized through intramuscular injection of a cocktail of ketamine, xylazine and atropine. Six animals were inoculated intranasally with 107 PFU of oseltamivir-resistant mutant, A/GN/1820, or oseltamivir-sensitive wild-type virus, A/KR/01, in 1 ml sterile phosphate-buffered saline (PBS). Two control animals were mock-inoculated with sterile PBS.
at −80 ◦ C and for histological study, those were fixed in 10% neutralbuffered formalin until processing. Virus replication in the lungs, nasal turbinates and major organs were measured by plaque assays in MDCK cells. The remaining animals were monitored daily for clinical signs, weights and temperature for 14 days. A scoring system of 0–3 was used to assess the activity of inoculated ferrets and a relative inactivity index (RII) was calculated as described previously (Reuman et al., 1989; Zitzow et al., 2002). Nasal washes were collected every other day for 9 days after inoculation and the virus titer was determined in MDCK cells. 2.5. Pathogenicity in mice Female 6- to 7-week-old BALB/c mice (Orient Bio, South Korea) were anesthetized with isoflurane and inoculated intranasally with 50 l of A/GN/1820 or A/KR/01 virus. The 50% mouse infectious dose (MID50 ) and 50% mouse lethal dose (MLD50 ) were calculated by inoculating eight mice per group with serial 10-fold dilutions, from 10◦ to 106 PFU, of virus. After 3 days, lungs of three mice from each group were collected after euthanasia and the viral titers were measured in MDCK cells for the calculation of MID50 by the method of Reed and Muench (Reed and Muench, 1938). The remaining five mice were monitored daily for body weight change and mortality for 14 days. To determine the pathogenicity and replication fitness of the virus, six mice were inoculated with 105 PFU of each virus and three were euthanized on days 3 and 6 p.i. Systemic organs were collected aseptically and frozen on dry ice immediately for virus titration and fixed in 10% neutral-buffered formalin for histological analysis. 2.6. Histological and immunohistochemical investigation Histopathological analyses of animal tissues were performed as described previously (Kwon et al., 2010, 2011). In short, after fixation in 10% neutral-buffered formalin, tissues were routinely processed and embedded in paraffin. The paraffin blocks were cut into 5 m-thick sections and stained with hematoxylin and eosin (H&E) for histopathological analysis. For the evaluation of influenza viral antigen expression, tissues were processed for immunohistochemical staining with influenza A specific antibodies. A monoclonal antibody (Novus Biologicals, Littleton, CO, USA) against the nucleoprotein of influenza A virus and goat polyclonal anti-influenza A antibody (Millipore, Billerica, MA, USA) were applied for the tissues of ferret and mouse, respectively. Then the sections were treated with biotinylated anti-mouse and anti-goat IgGs (Dako, Glostrup, Denmark), respectively. The slides were then applied with alkaline phosphatase conjugated streptavidin (Merck KGaA, Darmstadt, Germany) and were visualized by a solution of red substrate (Roche Diagnostics GmbH, Mannheim, Germany). The sections were lightly counterstained with Mayer’s hematoxylin and mounted with aqueous mount (Dako, Glostrup, Denmark). 2.7. Hemagglutination Inhibition assays Pre-exposure sera were collected before virus inoculation and post-exposure sera were collected at day 21 p.i. from ferrets and at day 14 p.i. from mice and H1-specific antibodies were evaluated by HI assays using 0.5% turkey red blood cells as described previously (Kwon et al., 2010).
2.4. Monitoring and collection of ferret specimens 3. Results Three infected ferrets and one mock-infected ferret were euthanized at day 3 post inoculation (p.i.) and tissue specimens (lung, trachea, nose, spleen, kidney, liver, heart, intestine, and brain) were collected for viral titration and histopathological analysis. For viral titration, specimens were immediately frozen on dry ice and stored
3.1. Sequence analysis of two viruses To evaluate the similarity of two viruses at molecular level, gene sequences of 8 fragments of each virus were analyzed. When
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Table 1 Phenotypic and genotypic characteristics of oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus. NA enzyme inhibition IC50 a (nM)
Virus
A/Korea/01/2009 A/Gyeongnam/1820/2009 a
Amino acid at NA 275
Oseltamivir carboxylate
Zanamivir
1.18 359.4
0.42 0.78
His Tyr
IC50 value: 50% inhibitory concentration (nM) of duplicate assays.
Table 2 Clinical signs of oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus infected ferrets. Virus
Sneezing (no/total)
Nasal discharge (no/total)
Lethargy (RII)
Weight loss (%)
Temperature rise (◦ C)
A/Korea/01/2009 A/Gyeongnam/1820/2009
3/3 1/3
2/3 2/3
1.8 1.5
5.8 3.9
1.2 1.8
compared with the sequences of A/KR/01 virus, A/GN/1820 virus had a total of 11 amino acid changes in the PB2 (L154I), PB1 (V645I), PA (K142M, K298E and S632A), HA (S220T), NA (N248D, H275Y, R319S and I467V) and NS (I123V) genes. As far as we know these changes were not known to be related with virulence or antiviral drug resistance except for H275Y in the NA gene. 3.2. In vitro sensitivity of viruses to NA inhibitors For the assessment of the in vitro sensitivity to NA inhibitors of A/GN/1820 and A/KR/01 virus, we measured NA activity using oseltamivir carboxylate and zanamivir and determined IC50 values. The IC50 value of A/GN/1820 virus against oseltamivir was about 300-fold higher than that of A/KR/01, however the sensitivity to zanamivir was comparable (Table 1). 3.3. Pathogenicity of viruses in ferrets Ferrets inoculated with oseltamivir-resistant, A/GN/1820, or oseltamivir-susceptible, A/KR/01, virus showed mild lethargy, infrequent sneezing and nasal discharge. All ferrets lost minimal weight, and body temperature rose at 1 or 2 day p.i. (Table 2). At day 3 p.i., A/GN/1820 virus replicated to high titer in the upper respiratory tract (nasal turbinates), while only two of three ferrets showed low levels of virus replication in the lower respiratory tract (lungs). No virus was identified from extrapulmonary organs of ferrets by plaque assays. In contrast, A/KR/01 virus replicated to high titer in the nasal turbinates and lungs, and to low titer in brain of ferrets (Table 3). In the nasal wash, both viruses replicated efficiently for 5 days with some differences in virus replication pattern on 5 day p.i. (Fig. 1). Serum HA antibodies against both viruses were elevated to 1:640 to 1:1280 at day 21 p.i. by HI assays. Histopathologically, two viruses caused similar microscopic lesions in the lungs of ferrets at day 3 p.i. Mild to moderate bronchopneumonia and severe interstitial pneumonia by thickening of the alveolar septa with extensive inflammatory cell infiltrates were detected and perivascular lymphoid tissue hyperplasia around the
Fig. 1. Viral titers of nasal washes from ferrets inoculated with oseltamivirsusceptible or oseltamivir-resistant pandemic (H1N1) 2009 influenza virus. Virus titers were determined every other day over 9 days post inoculation (p.i.) from nasal washes of ferrets inoculated with each virus. Error bars indicate the standard deviations of viral titers of three ferrets. Detection limit: 1.5 log10 PFU/ml.
blood vessels was found (Fig. 2A and B). We assessed influenza A viral antigen expression by the method of immunohistochemistry and both viruses showed similar pattern. Bronchial and bronchiolar epithelial cells, inflammatory cells in the bronchiolar lumen were stained clearly (Fig. 2C and D). We could detect viral antigen frequently in cells located in the thickened alveolar wall and pneumocytes. Peribronchial glands with severe peribronchitis were also infected by both viruses (Fig. 2E and F). Influenza A viral antigen was not detected in any other extrapulmonary tissues in ferrets inoculated with oseltamivir-resistant or oseltamivir-susceptible pandemic H1N1 influenza virus. 3.4. Pathogenicity of viruses in mice To evaluate pathological changes in mice we inoculated mice with serial 10-fold dilutions of each influenza virus. 104 PFU or more amounts of A/GN/1820 virus-infected mice showed weight
Table 3 Virus titers in organs of ferrets inoculated with oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus. Virus
Virus titer (mean log10 PFU ± SD/g)a Respiratory tract
A/Korea/01/2009 A/Gyeongnam/1820/2009
Brain
Nasal turbinates
Lung
7.5 ± 0.2 6.6 ± 0.1
6.7 ± 0.4 3.7/4.0/–
Trachea b
ND 3.0 ± 0.9
Olfactory bulb c
2.0/3.6/– –/–/–
Anterior part 3.9/–/– –/–/–
a Three ferrets were euthanized at day 3 post infection for viral titration. When viruses were recovered from all three ferrets, mean titers are presented. When viruses were not recovered from all animals, individual titers are shown. b ND: not done. c Virus not detected (detection limit: 1.5 log10 PFU/g of tissue).
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Fig. 2. Histopathologic changes and viral antigen expression in the lungs of inoculated with oseltamivir-susceptible or oseltamivir-resistant pandemic (H1N1) 2009 influenza virus. Ferrets were inoculated with 107 PFU of A/KR/01 (A, C, and E) or A/GN/1820 (B, D, and F) intranasally and were euthanized on day 3 p.i. and the tissues were processed for Hematoxylin and Eosin (A and B) and immunohistochemical staining (C–F). (A and B) Prominent alveolar thickening with extensive inflammatory cell infiltrates and suppurative exudates in the bronchiolar lumen. Magnification, 50×. (C and D) Extensive viral antigens (red stain) were detected in the bronchial epithelial cells (arrows). Magnification, 100×. (E and F) Viral antigens were detected in the peribronchial glands (arrowheads). Magnification, 200×.
loss by 7–8 day p.i. and the MID50 was 101.75 PFU. However, all mice survived during the period of the experiment at the highest dose tested (MLD50 > 106 PFU). 105 PFU or more amounts of A/KR/01 virus-infected mice also showed weight loss by 7 day p.i. and the MID50 was 101.5 PFU and all mice survived like A/GN/1820 virus. At day 3 and 6 p.i., both viruses replicated to high titer in the lungs of mice. A/GN/1820 virus replicated in trachea moderately and one of three mice had low titer of virus in the nose at 3 day p.i. (Table 4). No virus was detected in extrapulmonary organs. A/GN/1820 or A/KR/01 virus-infected mouse lung tissues showed mild to moderate multifocal necrotizing bronchiolitis and interstitial pneumonia. Prominent alveolar thickening with extensive inflammatory infiltrates was detected and perivascular cuffing with accumulation of lymphocytes around the blood vessels was found at day 3 p.i. (Fig. 3B and C). Positive staining by immunohistochemistry in tissues of mice was confined to lung and trachea. Viral antigen was detected extensively in bronchiolar epithelial cells,
pneumocytes and cells in the thickened alveolar wall (Fig. 3D and E). We could find several positive viral antigen signals in the tracheal epithelial cells (Fig. 3F), but not in any other extrapulmonary tissues. 4. Discussion During the 2009 H1N1 pandemic anti-influenza drugs were used extensively for the treatment and prophylaxis of influenza virus infection in South Korea. Korea CDC identified 11 cases of oseltamivir-resistant influenza virus among patients who had influenza that was refractory to antiviral treatment in South Korea (Shin et al., 2011; Yi et al., 2010). Because three of the 11 cases were fatal it was needed to assess the characteristics of the antiviralresistant H1N1pdm09 virus to compare the pathogenicity with antiviral-sensitive counterpart in animal models. In this study, we investigated the mammalian pathogenicity of oseltamivir-resistant H1N1pdm09 virus, A/GN/1820, which was
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Table 4 Virus titers in respiratory organs of mice inoculated with oseltamivir-susceptible and -resistant pandemic (H1N1) 2009 influenza virus. Virus
Virus titer (mean log10 PFU ± SD/g)a Lung
A/Korea/01/2009 A/Gyeongnam/1820/2009
Trachea
Nose
Day 3
Day 6
Day 3
Day 6
Day 3
Day 6
6.4 ± 0.4 7.2 ± 0.1
5.7 ± 0.3 6.7 ± 0.4
NDb 5.2 ± 0.2
ND 4.4 ± 0.1
2.9/2.7/–c 3.8/–/–
–/–/– –/–/–
a Three mice were euthanized at day 3 and 6 post infection for viral titration. When viruses were recovered from all three mice, mean titers are presented. When viruses were not recovered from all animals, individual titers are shown. b ND: not done. c Virus not detected (detection limit: 1.5 log10 PFU/g of tissue).
isolated from lethal case during 2009 H1N1 pandemic in South Korea. When compared with A/KR/01 virus which was the first isolate in South Korea in 2009, the amino acid sequences of A GN/1820 virus were changed at 11 sites in the PB2, PB1, PA, HA, NA and NS genes. However, we couldn’t find the changes related with increasing the virulence or antiviral drug resistance except for the H275Y mutation in the NA gene. Genotypic and phenotypic assay showed that this virus had H275Y mutation in NA gene and the IC50 value was about 300 times higher than that of sensitive counterpart A/KR/01 however A/GN/1820 virus showed sensitivity to zanamivir. To evaluate the fitness of oseltamivir-resistant mutant virus which caused mortality in human, we performed in vivo experiment using ferrets and mice and compared the results with those of antiviral-susceptible wild-type virus. Our study revealed that oseltamivir-resistant H1N1pdm09 virus isolated in South Korea replicated to similar levels with wildtype virus in upper respiratory tract however this mutant virus showed less efficient replication pattern in the lower respiratory tract of ferrets. Among three ferrets inoculated with A/GN/1820 virus, only two showed low level of virus replication in the lungs whereas all three A/KR/01 virus-inoculated ferrets showed high level of virus replication in lungs. Although both viruses caused similar clinical changes and histopathological pulmonary lesions in ferrets, oseltamivir-resistant mutant virus showed reduced
viral replication in ferret lungs. In mouse model oseltamivirresistant virus showed no significant difference in pathogenicity when compared with oseltamivir-susceptible virus however it required 10 times much amount of virus to cause weight loss. Immunohistochemical analysis provides valuable information on the pathogenesis of influenza virus. We could identify the replication sites and cell types in tissues of influenza virus infected animals. Although A/GN/1820 virus replicated less efficiently than A/KR/01 virus in ferrets lungs, the target cells were very similar. Both viruses replicated in bronchial/bronchiolar epithelial cells, interstitial macrophages, pneumocytes and peribronchial glands of ferrets. In mice, these viruses replicated mainly in bronchiolar epithelial cells, pneumocytes, interstitial inflammatory cells and tracheal epithelial cells. Chronological analysis showed that A/GN/1820 virus replicated highly in the mouse lungs from day 1 to day 7 p.i. without previous adaptation (data not shown). This result indicated that the pandemic H1N1 viruses whichever oseltamivir-resistant or oseltamivir-susceptible, could infect mice more efficiently than seasonal influenza viruses. Although this mutant virus was isolated from lethal infant case, the pathogenic properties in mammalian models showed less reduced viral fitness when compared with wild-type virus. The viral fitness, pathogenicity and transmission of several strains of the oseltamivir-resistant pandemic (H1N1) 2009 influenza virus have been studied in vitro and/or in vivo and the results have
Fig. 3. Representative histopathology and immunohistochemical results in respiratory organs from mice inoculated with oseltamivir-susceptible or oseltamivir-resistant pandemic (H1N1) 2009 influenza virus. Tissues were removed on 3 or 6 day p.i. and were processed for H&E (A–C) and immunohistochemical staining (D–F). (A) Mockinfected mouse lung showing normal histology. Magnification, 100×. (B and C) Lungs of A/GN/1820 virus-infected mice show prominent alveolar thickening with extensive inflammatory infiltrates (B) and perivascular cuffing (C) at day 6 p.i. Magnification, 100×. (D and E) Immunohistochemical staining of A/KR/01 (D) and A/GN/1820 (E) virus infected mouse lung at day 3 p.i. Influenza antigens were detected in the bronchiolar epithelial cells (arrows) and pneumocytes (arrowheads). Magnification, 200×. (F) Viral antigen was detected in A/GN/1820 virus-infected mouse tracheal epithelial cells (arrows). Magnification, 200×.
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shown variable pathogenic properties depending on the isolates used when compared with the oseltamivir-sensitive counterparts (Duan et al., 2010; Hamelin et al., 2010; Kiso et al., 2010; Memoli et al., 2011; Seibert et al., 2010). Previous studies revealed that the oseltamivir-resistant viruses possess lower or equivalent transmissibility in ferrets depending on the isolates (Duan et al., 2010; Kiso et al., 2010; Seibert et al., 2010). However we do not know if the Korean mutant virus can be transmissible in ferrets because we could not perform transmission experiments and this is the limitation in our study and needs further study. The extensive usage of oseltamivir may trigger the emergence of NA inhibitor-resistant mutants which possess efficient replication fitness, virulence and transmissibility in humans. In South Korea, most of the oseltamivir-resistant viruses were identified in patients with underlying diseases and caused death in 30 per cent (Yi et al., 2010). Therefore it is very important to find antiviralresistant mutant viruses through national surveillance system and it needs more caution when treat patients with underlying disease. Conflict of interest There are no actual or potential conflicts of interest. Acknowledgements This study was supported by the Intramural Research Program (2010-N43001-00) of the Korea National Institute of Health, Korea Centers for Disease Control and Prevention. References Dawood, F.S., Jain, S., Finelli, L., Shaw, M.W., Lindstrom, S., Garten, R.J., Gubareva, L.V., Xu, X., Bridges, C.B., Uyeki, T.M., 2009. Emergence of a novel swine-origin influenza a (H1N1) virus in humans. N. Engl. J. Med. 360, 2605–2615. Duan, S., Boltz, D.A., Seiler, P., Li, J., Bragstad, K., Nielsen, L.P., Webby, R.J., Webster, R.G., Govorkova, E.A., 2010. Oseltamivir-resistant pandemic H1N1/2009 influenza virus possesses lower transmissibility and fitness in ferrets. PLoS Pathog. 6, e1001022. Hamelin, M.E., Baz, M., Abed, Y., Couture, C., Joubert, P., Beaulieu, E., Bellerose, N., Plante, M., Mallett, C., Schumer, G., Kobinger, G.P., Boivin, G., 2010. Oseltamivirresistant pandemic A/H1N1 virus is as virulent as its wild-type counterpart in mice and ferrets. PLoS Pathog. 6, e1001015.
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