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Journal of General Virology (2014), 95, 779–786 DOI 10.1099/vir.0.061721-0 The PB2 E627K mutation contributes to the high polymerase activity and enhanced replication of H7N9 influenza virus Hong Zhang,13 Xuyong Li,23 Jing Guo,2 Li Li,1 Chong Chang,1 Yuanyuan Li,2 Chao Bian,3 Ke Xu,1 Hualan Chen2 and Bing Sun1,3 1 Correspondence Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China Bing Sun [email protected] Hualan Chen 2 State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China [email protected] 3 State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China Received 14 November 2013 Accepted 4 January 2014 Human infection by H7N9 influenza virus was first identified in China in March 2013. As of 12 August 2013, a total of 135 documented cases with 44 fatalities had been reported. Genetic and laboratory analyses of the novel H7N9 viruses isolated from patients indicate that these viruses possess several polymerase gene mutations previously associated with human adaptation and potential pandemic capabilities. However, the function of these mutations in the emergence and pathogenicity of the viruses is not well known. In this study, we demonstrate that the PB2 E627K mutation, which occurs in over 70 % of the H7N9 patient isolates, promotes the replication of H7N9 virus by enhancing PB2 polymerase activity and enhances virulence in mice. Our results show the PB2 E627K mutation has played an important role in this H7N9 influenza outbreak and in the pathogenicity of the H7N9 virus. INTRODUCTION On 29 March 2013 the Chinese Center for Disease Control and Prevention confirmed the first case of human infection with H7N9 influenza A virus (Gao et al., 2013b). As of 12 August 2013, 135 cases with 44 fatalities had been confirmed, causing worldwide concern (Zhang et al., 2013). Patients infected with H7N9 viruses have a rapidly progressive pneumonia, leading to respiratory failure and acute respiratory distress syndrome (Gao et al., 2013a). Phylogenetic analysis suggests that the novel H7N9 virus is a triple reassortant and that its viral genes are of avian origin. The surface glycoprotein haemagglutinin (HA) was derived from the H7N3 virus from domestic ducks in Zhejiang, whereas the neuraminidase (NA) was derived from the wild bird H7N9 virus in South Korea. All six internal genes show high similarity to the poultry H9N2 virus (Gao et al., 2013b; Kageyama et al., 2013). Human 3These authors contributed equally to this work. The GenBank/EMBL/DDBJ accession numbers for the H7N9 PB2 influenza virus sequences are AGK84850, AGK84856, AGK84859, AGO51410, AGO51398, AGO51442, AGJ73498, AGL44433, AGI60293, AGN69469, AGN69457, AGJ51961, and AGM16245. 061721 G 2014 SGM infections with H7N9 virus have not been reported previously, and animal infections with H7N9 viruses had not been detected in China before this outbreak. The influenza virus is a continuous threat to human health. In addition to the annual seasonal epidemic, the influenza virus occasionally causes pandemics. During the past century, several pandemics have occurred, including 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 2009 (pH1N1); all the pandemic viruses bear HA and NA genes of avian or pig origin (Liu et al., 2013). Owing to the low fidelity of the viral RNA-dependent polymerase, it is easy for viruses to acquire adaptive mutations that allow transmission to hosts of different species. In modern times, direct human infections with avian influenza viruses occur only sporadically, including with the H7N2 (Ostrowsky et al., 2012), H7N3 (Skowronski et al., 2006; Tweed et al., 2004), H7N7 (Fouchier et al., 2004), H9N2 (Blair et al., 2013; Peiris et al., 1999) and H5N1 (de Jong et al., 1997) subtypes. Most human infections result in mild illness and conjunctivitis, except for the H5N1 subtype, which is associated with greater than 50 % mortality. Patients infected with the H7N9 subtype have been detected in over 10 provinces of China, and most experienced a severe clinical syndrome. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 16:01:40 Printed in Great Britain 779 H. Zhang and others At this time, no investigations have revealed evidence of sustained spread of this virus in humans. However, the possibility of limited human-to-human spread cannot be excluded in a few small clusters of human H7N9 virus infections (Qi et al., 2013). Further analysis of the H7N9 gene segments has shown the presence of signature amino acids associated with adaptation to the human host and with virulence, such as the Q226L mutation in HA, which is associated with increased binding to mammalian-like receptors in the human upper airway, and the E627K and D701N mutations in PB2, which are associated with enhanced replication and virulence (Kageyama et al., 2013; Li et al., 2005; Liu et al., 2013). During the past 2 months, many studies have shown that H7N9 viruses have a mixed a-2,3/a-2,6 receptor preference, which is regulated by the single Q226L mutation (Belser et al., 2013). The H7N9 viruses were transmitted through close contact between ferrets, but they were not well transmitted by droplets. However, one isolate of H7N9 from a patient in Anhui was demonstrated to be highly transmissible between ferrets by respiratory droplets (Zhang et al., 2013). The PB2 E627K mutation has been recognized as one of the most important mammalian adaptive markers. Since the beginning of the outbreak of highly pathogenic avian H5N1, the percentage of H5N1 isolates containing PB2 627K has increased. In this current outbreak of H7N9, a large percentage of the viruses isolated from patients contain 627K in PB2, whereas all the viruses isolated from poultry and from the environment contain 627E (Li et al., 2013; Shi et al., 2013). Thus, it is reasonable to suppose that the PB2 E627K substitution plays an important role in the transmission and virulence of the novel H7N9 virus. However, the role of PB2 627K in the emergence of H7N9 needs to be experimentally demonstrated. In this study, we tested the polymerase activity and replication kinetics of the H7N9 virus harbouring 627K/E in cell cultures. In addition, the pathogenicity of H7N9 was tested in mice by measuring body mass for two weeks postinfection and determining organ viral load in selected organs. We demonstrate that the PB2 E627K substitution contributes to the replication and pathogenicity of the H7N9 virus by regulating polymerase activity. RESULTS The PB2 E627K mutation was prevalent in human isolates of H7N9 virus Gene analysis of the H7N9 virus has shown that the new viruses contain some mutations associated with adaptation to human hosts, such as PB2 E627K. To evaluate the role of PB2 E627K in the emergence of H7N9, we collected all the sequences of H7N9 isolated from humans and avian hosts. The percentage of viruses containing the PB2 E627K mutation was calculated. The percentage of H7N9 patient isolates containing PB2 E627K is high, at 71.4 % [PB2 627K 780 was found in 10 of 14 human H7N9 isolates and only four isolates downloaded from the National Center for Biotechnology Information (NCBI) were PB2 627E], and the fatality rate was approximately 30 % (http://www.who. int/influenza/human_animal_interface/influenza_h7n9/Data_ Reports/en/). All of the 37 H7N9 viruses isolated from avian or environmental sources contain PB2 627E (Zhang et al., 2013). Together, these data suggest that the PB2 E627K mutation plays an important role in the occurrence of H7N9 from avian sources in human hosts. The PB2 E627K mutation contributes to high polymerase activity and enhanced replication of H7N9 To test the contribution of PB2 E627K to H7N9 polymerase activity, we performed a luciferase reporter assay. We used WSN (Influenza A/WSN/1933 (H1N1)) virus as a reference strain. The polymerase activity of H7N9 627K was approximately 20-fold higher than that of H7N9 627E in 293T cells (Fig. 1a). However, there was not as big a difference between the polymerase activity of H7N9 627K and 627E in the chicken embryo fibroblast (CEF) cell line DF-1 as that in mammalian cells (Fig. 1b). The polymerase activity pattern of PB2 627K/E in H7N9 was similar to the activity observed in H5N1 cases (Mänz et al., 2012). To further confirm the contribution of PB2 627K to H7N9 replication, we compared the replication kinetics of H7N9 viruses containing PB2 627K and 627E in the A549 human alveolar epithelial cell line and primary CEFs. The virus titre of H7N9 was approximately 100-fold higher than that of H7N9 PB2 K627E from 12 h onwards after infection in A549 cells. The viral titre of H7N9 PB2 K627E did not increase during the infection, suggesting that the virus is not well adapted to the A549 cell line (Fig. 1c). There was no apparent difference in viral titre between H7N9 and H7N9 PB2 627E in CEFs (Fig. 1d). Taken together, these results indicate that PB2 627K enhances polymerase activity and viral replication in mammalian cells. Polymerase activity of H7N9 with PB2 627K is higher than with PB2 627E and is capable of promoting virus replication at 33 ‡C Although the mechanism of how PB2 E627K exerts its effects is not yet clear, one possible function of PB2 627K is to facilitate replication of avian viruses in the human upper respiratory tract, which generally has a temperature of 33 uC. In contrast, the temperature of the avian intestinal tract is closer to 41 uC, a temperature at which PB2 627E facilitates efficient viral replication (Hatta et al., 2007; Steel et al., 2009). To address whether the PB2 E627K mutation influences polymerase activity at a lower temperature, we tested the polymerase activity of WSN and H7N9 viruses with PB2 627K or 627E at 33 uC in 293T cells. The polymerase activity of H7N9 was still relatively high and about 1.5-fold higher than that of WSN at 33 uC. It was obvious that the polymerase activity of H7N9 viruses with Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 16:01:40 Journal of General Virology 95 (a) 300 (b) 800 Relative polymerase activity Relative polymerase activity PB2 E627K promotes high polymerase activity of H7N9 50 PB 2 PB 2 H7N9 H7N9-PB2 K627E 6 4 2 24 36 48 60 Time post infection (h) K6 27 E SN 7N 9 SN H W H (d) 8 72 Viral titres (log10 EID50 ml–1) Viral titres (log10 EID50 ml–1) W PB 2 7N 9 SN W A549 (c) 12 0 K6 27 E 9 7N H PB 2 W K6 27 E SN 0 200 K6 27 E 100 400 9 150 600 7N 200 H 250 CEF H7N9 H7N9-PB2 K627E 8 7 6 5 4 12 48 60 24 36 Time post infection (h) 72 Fig. 1. Polymerase activity and replication kinetics of H7N9 in cell cultures. The polymerase activity of WSN, WSN 627E, H7N9 and H7N9 627E was tested in 293T cells (a) and DF-1 cells (b). Fifty nanograms each of PB1, PB2, PA and NP in pCAGGS; 100 ng of human pPolI-NP-luc or avian pPolI-NS-luc; and 10 ng of pRLSV40 (Promega) were co-transfected into 293T cells or DF-1 cells, which were harvested 24 h after transfection. All data were normalized to the activity of the WSN sample. Growth curves of H7N9 and H7N9 PB2 K627E were studied in A549 cells (c) and CEFs (d). Cells were infected with viruses at an m.o.i. of 0.01. At 12, 24, 48 and 72 h post-inoculation, the supernatants were harvested, and virus titres were determined in eggs. PB2 627K was about 60-fold higher than that of H7N9 viruses with PB2 627E (Fig. 2a). These data suggest that the H7N9 viruses may have the ability to replicate well in the human upper respiratory tract at 33 uC. To test the contribution of PB2 627K to viral replication at 33 uC, the replication kinetics of H7N9 were tested in A549 cells. As shown in Fig. 2b, compared with H7N9 PB2 K627E, H7N9 grew well in A549 cells at 33 uC. These data show that H7N9 is more adapted to mammalian cells than H7N9 PB2 K627E at 33 uC. PB2 627K contributes to the replication and transcription processes in mammalian cells As we have demonstrated that PB2 627K contributes to the high polymerase activity and enhanced replication of H7N9 viruses in mammalian cells, we further investigated whether the higher activity associated with PB2 627K was http://vir.sgmjournals.org related to an increase in transcription (mRNA) and/or replication (cRNA and vRNA synthesis) by using a quantitative PCR-based assay. H7N9 viruses harbouring PB2 627K had a higher transcription activity at 33 uC, as demonstrated by a .10-fold higher level of PB2 627K than PB2 627E mRNA. The cRNA of H7N9 viruses harbouring PB2 627K was about threefold higher than that of H7N9 viruses harbouring PB2 627E, although the vRNA level of H7N9 with PB2 627K was slightly higher than that of H7N9 with PB2 627E (Fig. 3b). Conversely, the H7N9 viruses containing PB2 627K had a higher replication and transcription activity at 37 uC, as demonstrated by an approximately twofold increase in the level of all three kinds of RNA of PB2 627K compared with that of PB2 627E (Fig. 3a). These data indicate that PB2 627K enhances polymerase activity by regulating the viral replication and transcription process in mammalian cells. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 16:01:40 781 H. Zhang and others (b) Viral titre (log10 EID50 ml–1) 200 150 100 50 8 H7N9 H7N9-PB2 K627E 6 4 2 48 60 24 36 Time post infection (h) 12 K6 27 E 7N H 72 H W 7N 9 SN PB 2 PB 2 W K6 27 E 9 0 SN Relative polymerase activity (a) Fig. 2. Polymerase activity and replication kinetics of H7N9 harbouring PB2 627K or 627E at 33 6C. (a) Polymerase activity of H7N9 and H7N9 627E in 293T cells at 33 6C. Fifty nanograms each of PB1, PB2, PA and NP in pCAGGS; 100 ng of human pPolI-NP-luc; and 10 ng of pRLSV40 (Promega) were co-transfected into 293T cells, which were harvested 24 h after transfection. All data were normalized to the activity of the WSN sample. (b) Replication kinetics of H7N9 harbouring PB2 627K or 627E at 33 6C. A549 cells were infected with viruses at an m.o.i. of 0.01. At 12, 24, 48 and 72 h post-inoculation, the supernatants were harvested and virus titres were determined in eggs. To further determine the pathogenicity and replication of H7N9 harbouring 627K or 627E in mammals, mice were infected with 106 median 50 % egg-infectious dose (EID50) of both viruses, and organs were harvested at 3 and 5 days post-infection. The body mass of mice infected with H7N9 decreased by approximately 20 %, while the body mass of mice infected with H7N9 PB2 K627E did not decrease (Fig. 4a). The replication of H7N9 and H7N9 PB2 K627E was 100 80 60 40 20 0 vRNA cRNA mRNA 33 °C 100 80 60 40 20 E 9 K6 H 2 PB H 7N 9 PB 9 7N 27 7N E 9 27 7N 2 K6 H 27 K6 H H 7N 9 PB 9 7N E 9 2 2 K6 H 27 7N E 9 7N H 27 H 7N 9 PB 2 K6 H E 9 7N E 27 H 9 PB 2 K6 H 7N 9 0 7N H (b) 120 PB Relative vRNA level vRNA cRNA mRNA 37 °C (a) 120 detected in nasal turbinates and lungs but not in other organs (brain, spleen and kidneys). The viral titres of H7N9 in nasal turbinates and lungs was significantly higher than the titres of H7N9 PB2 K627E (Fig. 4b, c). The replication of H7N9 was similar in nasal turbinates and the lung, whereas the viral titre of H7N9 PB2 K627E in nasal turbinates was lower than that in the lung. Our data suggest that PB2 627K promotes viral replication in mice, and H7N9 virus containing PB2 627K is better adapted to the upper and lower respiratory tracts than virus containing PB2 627E. Relative vRNA level PB2 627K enhances the replication of H7N9 in mice Fig. 3. Quantification of viral RNA levels of H7N9RNP complexes containing PB2 627K or 627E at 37 6C and 33 6C. 293T cells were co-transfected with expression plasmids encoding NP, PA, PB1 and PB2 together with pPolI-NA plasmid and incubated at (a) 37 6C or (b) 33 6C. Total cellular RNA was isolated 24 h post-transfection and was subjected to quantitative RT-PCR for segment 6 (NA gene) transcripts. All data were normalized to viral RNA, cRNA and mRNA of the H7N9 sample. Results are means±SD from three independent assays. 782 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 16:01:40 Journal of General Virology 95 PB2 E627K promotes high polymerase activity of H7N9 H7N9 0 ey n te rb in a as al tu n ey Ki dn Sp le e Lu Br ai n Organs N N as al tu rb in a ng 0 te 0 2 4 6 8 10 12 14 Time post inoculation (days) 1 Ki dn 60 2 2 Sp le e 80 4 Day 3 Day 5 3 Br ai n 100 6 ng 120 H7N9 PB2 K627E 4 Viral titres (log10 EID50 ml–1) 140 Day 3 (c) Day 5 8 Lu (b) H7N9 H7N9 PB2 K627E Viral titres (log10 EID50 ml–1) Body mass change (%) (a) Organs Fig. 4. Replication of H7N9 and H7N9 PB2 K627E in mice. (a) Body mass of mice infected with H7N9 and H7N9 PB2 K627E. Mice were inoculated with 106 EID50 of virus H7N9 or H7N9-PB2 K627E in a volume of 50 ml. Body mass was recorded for 14 days after infection. Virus titres of H7N9 (b) and H7N9 PB2 K627E (c) in mouse organs were determined. Nasal turbinates, lungs, spleen, kidneys and brain were harvested at 3 days and 5 days post-infection, and virus titres were determined in eggs. Dashed lines indicate the lower limit of detection. DISCUSSION The emergence of a new H7N9 avian influenza is a global health concern due to the severity of infection and associated mortality in humans. During the past several decades, human infections with other H7-subtype avian viruses have been reported, but most of them resulted in only mild illness and conjunctivitis, except for one death caused by H7N7 in The Netherlands (Fouchier et al., 2004). However, this is the first time that the N9 subtype has been reported in human infections. Genetic analysis has predicted that the viruses contain some mutations related to human adaptation, such as PB2 E627K, a hallmark of influenza virulence and transmission to humans. Therefore, in this study we experimentally clarified the contribution of PB2 627K to the replication and pathogenicity of H7N9. To evaluate the role of PB2 627K in the outbreak of H7N9, we collected all the PB2 sequences of human-isolated and avian or environment-isolated H7N9 from NCBI. The percentage of PB2 627K was higher in H7N9 isolated from patients, but there was no PB2 627K in the avian or environment-isolated H7N9. The evidence suggests that PB2 627K may play an important role in human H7N9 infection. However, the PB2 627K is not the only factor that determines polymerase activity and adaptation to humans. There may be other mutations in polymerase genes that promote the viral replication in H7N9, such as PB2 591K/R, PB2 701N (Gabriel et al., 2005; Mehle & Doudna, 2009; Yamada et al., 2010) and PB1 (473V and 598P) (Xu et al., 2012). Four H7N9 isolates in our collections contained PB2 627E: A/Nanjing/1/2013, A/Jiangsu/1/2013, A/Jiangsu/2/ 2013 and A/Zhejiang/DTID-ZJU01/2013. The isolate from Zhejiang harbours PB2 701N, which is predicted to complement the loss of PB2 627K. In contrast, the isolates from Nanjing and Jiangsu have PB2 591K and PB1 473V. In future, we should monitor the evolution of H7N9 in case these viruses acquire more adaptive mutations. http://vir.sgmjournals.org PB2 627K contributes to high polymerase activity in mammalian cells, but not in avian cells. Using a luciferase reporter assay, we demonstrated that PB2 627K promotes the polymerase activity of H7N9 in mammalian cells, but not in avian cells. In addition, viral titre studies in mice demonstrate that PB2 627K contributes to the enhanced replication of H7N9 in vivo. Many potential mechanisms of the high polymerase activity associated with PB2 627K have been proposed. Amino acid 627 lies on the surface of a polymerase PB2 subunit and is involved in the interaction with the host factor importin (Tarendeau et al., 2008). The differential use of importin-a isoforms governs cell tropism and host adaptation of influenza virus based on studies investigating the transmission of avian influenza H5N1 (Gabriel et al., 2008, 2011). Some studies have shown that the enhanced interaction between NP and PB2 mediated by PB2 627K contributes to the high polymerase activity in mammalian cells but not in avian cells (Labadie et al., 2007; Rameix-Welti et al., 2009). Some studies suggested that the adaptive mutation PB2 E627K was mediated by an inhibitory or stimulatory factor in host cells (Mehle & Doudna, 2008; Moncorgé et al., 2010). The crystallography indicated that the amino acid PB2 627 was in the Cterminal RNA binding domain and the PB2 627K had higher RNA binding activity than the PB2 627E (Kuzuhara et al., 2009). More experiments should be performed to test this mechanism in the H7N9 virus. The stability of influenza virus is temperature dependent. To cause a pandemic, a virus needs to maintain a stable state and replicate well at a lower temperature (33 uC), which is the approximate temperature of the human upper respiratory tract. PB2 627K improves polymerase activity at lower temperatures and is thought to confer stability on viruses, which allows the virus to replicate better at the lower temperature (Steel et al., 2009). We tested the polymerase activity of PB2 627K at 33 uC. H7N9 harbouring PB2 627K had relatively high polymerase activity at 33 uC. The viral titre Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 16:01:40 783 H. Zhang and others in nasal turbinates also indicated that H7N9 is well adapted to the upper respiratory tract. In addition, PB2 627K may improve polymerase activity by regulating the transcription and replication processes at different temperatures. 37 uC, respectively. Culture supernatant was collected at the indicated time points post-infection and titrated in eggs. The growth data shown are the average results of three independent experiments. Our study characterized the H7N9 virus (harbouring PB2 627K) and found that it possessed higher polymerase activity and improved replication in mammalian cells when compared to H7N9 harbouring PB2 K627E. More importantly, PB2 627K promoted the replication of H7N9 in nasal turbinates and lungs in mice and can cause illness. Our data indicate that the PB2 627K plays an important role in the outbreak and pathogenicity of H7N9. PA and NP; 100 ng of pPolI-NP-luc; and 10 ng of pRLSV40 (Promega) were co-transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection at 37 uC and 33 uC. Luciferase assays were performed using the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s protocol. The firefly and Renilla luciferase activities were measured using a microplate luminometer (Veritas). The ratio of firefly luciferase activity to Renilla luciferase activity was calculated to represent the efficiency of the transcription/replication of the viral-like reporter RNA. All experiments were performed in triplicate. Results are presented as the mean±SD. METHODS Facility. All experiments with live H7N9 viruses were conducted within the enhanced animal biosafety level 3 (ABSL3+) facility in the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, approved for such use by the Ministry of Agriculture of China and the China National Accreditation Service for Conformity Assessment. Plasmids. PB1, PB2, PA and NP from A/Anhui/1/2013 (H7N9) were commercially synthesized and cloned into the vector pCAGGS with restriction enzyme EcoRI (Takara). Viral cDNAs from the A/WSN/33 (H1N1) virus were kindly provided by Professor Hans Klenk (Marburg University, Germany) and were cloned into the vector pCAGGS (kindly provided by Dr Jun-ichi Miyazaki, Osaka University, Japan). The pPolI-NP-luc and pPolI-NA were also provided by Professor Hans Klenk. The avian pPolI-NP-luc was constructed by replacing the human pPolI-NP-luc promoter by the avian promoter. Cells. Human embryonic kidney 293T cells and immortalized CEFs (DF-1) were purchased from the ATCC and maintained in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10 % FBS (Gibco) plus penicillin and streptomycin. Alveolar basal epithelial cells (A549) were maintained in F-12K Nutrient Mixture (Gibco) containing 10 % FBS plus antibiotics. Madin–Darby canine kidney cells were grown in minimum essential medium with Eagle’s salts containing 4 % FBS, 4 mM L-glutamine, and antibiotics. Reverse genetics. An eight-plasmid reverse genetics system was used to generate H7N9 and H7N9-PB2 K627E viruses. As described previously, cDNA from the human-infecting H7N9 influenza virus A/Anhui/1/2013 (AH1) was inserted into the bidirectional transcription vector pBD. We introduced the mutation PB2 K627E into the AH1-PB2 plasmid by site-directed mutagenesis with the QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The plasmids used for virus rescue and the genes from the rescued viruses were fully sequenced to confirm the absence of unwanted mutations. Virus rescue was performed as previously described (Li et al., 2005). Briefly, 293T cells were co-transfected with 0.5 mg of each of the eight plasmids mixed with 10 ml Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions. Eight hours later, the DNA-transfection mixture was replaced by Opti-MEM (Gibco). The supernatant was harvested and injected into 10-day-old specific pathogen-free embryonated eggs for virus propagation after 48 h. The rescued virus was detected by haemagglutination assay. Luciferase reporter assay. Fifty nanograms each of PB1, PB2, Strand-specific real-time RT-PCR assay. Fifty nanograms each of PB1, PB2, PA and NP in pCAGGS and 100 ng of pPolI-NA (provided by Professor Hans Klenk) were co-transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested 24 h after transfection at 37 uC and 33 uC, and the total cellular RNA was extracted with TRIzol RNA isolation reagents (Invitrogen). RNA (0.5 mg of each sample) was reverse-transcribed by using the strand-specific tagged primers (Kawakami et al., 2011) for the NA gene using a ReverTra Ace qPCR RT kit (Toyobo) according to the manufacturer’s instructions. Realtime quantitative PCR (qPCR) was performed with SYBR Green Realtime PCR Master Mix (Toyobo) according to the manufacturer’s instructions on an ABI PRISM 7900HT. The qPCR cycle conditions were 95 uC for 10 min followed by 40 cycles of 95 uC for 15 s and 60 uC for 1 min. The vRNA, cRNA and mRNA levels were expressed relative to GAPDH mRNA as ratios. Mouse infection. To evaluate the virulence of H7N9 influenza virus in a mammalian host, two groups (n511) of 6–7-week-old female BALB/c mice (Vital River) were inoculated with 106 EID50 of the H7N9 virus or the H7N9-PB2 K627E virus in a volume of 50 ml. Three mice were euthanized at 3 and 5 days post-inoculation, respectively, and their nasal turbinates, lungs, spleen, kidneys and brain were suspended in 1 ml of cold sterile PBS and subsequently homogenized for viral titration. The other mice were weighed and observed for signs of disease for 2 weeks. ACKNOWLEDGEMENTS This work was supported by grants from the National 973 key project (2013CB530504), and the National 863 project (2012AA02A404, 2012AA020103), the National Science and Technology Major Project (2012ZX10002-007-003, 2013ZX10004-101-005, 2013ZX10004003-003), grants from the National Natural Science Foundation of China (31030029, 31230024, 81201280), a grant from CAS Key Project (KSZD-EW-Z-002-3) and a grant from the Science and Technology Commission of Shanghai Municipality (12ZR1435000). Finally this work is the following study of European Project SP5BCT-2006-044161 (to B. S.). This work was also supported by grants from the Ministry of Science and Technology (2012ZX10004214) (to H. C.). REFERENCES Viral growth kinetics. Viruses were inoculated into A549 or CEF monolayers at an m.o.i. of 0.01. One hour after infection, the cells were replaced with fresh Opti-MEM and incubated at 33 uC and 784 Belser, J. A., Gustin, K. M., Pearce, M. B., Maines, T. 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