Adaptive amino acid substitutions enhance the virulence of an H7N7 avian influenza virus isolated from wild waterfowl in mice.

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Adaptive amino acid substitutions enhance the virulence of an H7N7 avian influenza virus isolated from wild waterfowl in mice Qiang Chen a,d,1, Zhijun Yu b,c,1, Weiyang Sun b,1, Xue Li b,e,1, Hongliang Chai a,1, Xiaolong Gao b, Jiao Guo b, Kun Zhang b,c, Na Feng b, Xuexing Zheng b, Hualei Wang b, Yongkun Zhao b,f, Chuan Qin c, Geng Huang b, Songtao Yang b, Jun Qian b,f, Yuwei Gao b,f, Xianzhu Xia b,c,f,*, Tiecheng Wang b,f,**, Yuping Hua a,*** a

College of Wildlife Resources, Northeast Forestry University, Harbin 150040, People’s Republic of China Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, Changchun 130122, People’s Republic of China c Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, People’s Republic of China d Liaoning Medical University, Jinzhou 121001, People’s Republic of China e Changchun Institute of Biological Products, Changchun 130122, People’s Republic of China f Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, People’s Republic of China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 October 2014 Received in revised form 13 February 2015 Accepted 18 February 2015

Although H7N7 AIVs primarily circulate in wild waterfowl, documented cases of human infection with H7N7 viruses suggest they may pose a pandemic threat. Here, we generated mouse-adapted variants of a wild waterfowl-origin H7N7 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the adapted variants were reduced >5000-fold compared to the parental virus. Mouseadapted variants viruses displayed enhanced replication in vitro and in vivo, and acquired the ability to replicate in extrapulmonary tissues. These observations suggest that enhanced growth characteristics and modified cell tropism may increase the virulence of H7N7 AIVs in mice. Genomic analysis of the adapted variant viruses revealed amino acid changes in the PB2 (E627K), PB1 (R118I), PA (L550M), HA (G214R), and NA (S372N) proteins. Our results suggest that these amino acid substitutions collaboratively enhance the ability of H7N7 virus to replicate and cause severe disease in mammals. ß 2015 Elsevier B.V. All rights reserved.

Keywords: Wild waterfowl Avian influenza virus H7N7 Mice Adaptation

* Corresponding author at: The Military Veterinary Institute, Academy of Military Medical Science, PLA 666 Liuyingxi Street, Changchun 130122, People’s Republic of China. Tel.: +86 431 8698 5516; fax: +86 431 8698 5516. E-mail address: [email protected] (X. Xia). ** Corresponding author at: The Military Veterinary Institute, Academy of Military Medical Science, PLA 666 Liuyingxi Street, Changchun 130122, People’s Republic of China. Tel.: +86 431 8698 5517; fax: +86 431 8698 5517. E-mail address: [email protected] (T. Wang). *** Corresponding author. Tel.: +86 18249095870; fax: +86 451 82190390. E-mail address: [email protected] (Y. Hua). 1 These authors contributed equally to the results of this study. http://dx.doi.org/10.1016/j.vetmic.2015.02.016 0378-1135/ß 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Chen, Q., et al., Adaptive amino acid substitutions enhance the virulence of an H7N7 avian influenza virus isolated from wild waterfowl in mice. Vet. Microbiol. (2015), http://dx.doi.org/10.1016/ j.vetmic.2015.02.016

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1. Introduction Wild waterfowl are the natural reservoir of avian influenza viruses (AIVs) (Webster et al., 1992). While AIVs typically replicate efficiently in their natural hosts, there is evidence that AIVs can infect mammals, including dogs, cats, plateau pikas, rhesus macaques, and humans (Cheng et al., 2014b; Shinya et al., 2012; Songserm et al., 2006; Yu et al., 2014a; Zhang et al., 2013a, 2013b). Some AIVs are reported to be capable of transmission between mammals, suggesting that AIVs may possess pandemic potential (Belser et al., 2013; Gao et al., 2009; Zhang et al., 2013c). H7N7 viruses circulate widely in areas including North America (Spackman et al., 2008) and Europe (Slavec et al., 2012), as well as southern China (Lam et al., 2013), Australia (Bulach et al., 2010), and New Zealand (Bulach et al., 2010). Zoonotic transmission of H7 influenza viruses to humans has been suggested by reports of significantly elevated titers of H7-specific antibodies among poultry workers in Italy (Di Trani et al., 2012), and documented cases of human H7N7 infection in the Netherlands in 2003 and H7N9 infection in China in 2013 (Gao et al., 2013; Koopmans et al., 2004). A previous report found that ferrets inoculated by the ocular aerosol route with an avian H7N7 virus were capable of transmitting the virus to naı¨ve animals in direct-contact or respiratory-droplet models (Belser et al., 2014). Taken together, reports of human infection with H7N7 AIVs and a lack of pre-existing immunity against these viruses in humans suggest that H7N7 AIVs may pose a pandemic threat. Therefore, the molecular features involved in the mammalian adaptation of H7N7 AIVs should be further studied. While mice are not a natural host for influenza and viruses that cause severe disease in mice usually show little virulence in other mammals, they are a conventional animal model in which to study the pathogenesis of AIVs (Belser et al., 2007, 2013; Belser and Tumpey, 2013; Cheng et al., 2014a; Gabriel et al., 2005; Hatta et al., 2001; Li et al., 2005, 2014; Song et al., 2009; Tumpey et al., 2002). To identify the possible changes that are associated with the adaptation of H7N7 AIV to mammals, we serially passaged a wild waterfowl-origin H7N7 virus in mice. After sequential passage of an H7N7 virus in mice, we obtained two viruses which displayed increasing virulence and enhanced replication kinetics in vitro and in vivo. Multiple amino acid substitutions involved in the adaptation of H7N7 avian influenza virus in mice. While the adaptive mutations identified in this study may contribute to mammalian adaptation of wild bird-origin H7N7 AIVs, the effects of these mutations on mammalian pathogenicity need to be studied in the future works.

2. Materials and methods 2.1. Facilities Studies with H7N7 AIVs were conducted in a biosecurity level 3 laboratory approved by the Military Veterinary Research Institute of the Academy of Military Medical Sciences. All animal studies were approved by the

Review Board of Military Veterinary Research Institute of the Academy of Military Medical Sciences. 2.2. Cells and virus Madin–Darby canine kidney (MDCK) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM glutamine, 5% fetal calf serum (FCS), 100 IU/ml penicillin, and 100 mg/ml streptomycin. 293T cells were cultured in DMEM supplemented with 2 mM glutamine, 10% FCS, 100 IU/ml penicillin, and 100 mg/ml streptomycin. The H7N7 virus A/Lesser White-fronted Goose/HuNan/412/2010 (H7N7) (abbreviated as 3Y) was isolated from a lesser white-fronted goose in Hunan province, China. This virus has a monobasic cleavage site (NVPEIPKGR/G). Stock viruses were grown in the allantoic cavities of 10-day-old chicken eggs for 48 h at 37 8C, and aliquots were stored at 80 8C until used. The GenBank accession numbers corresponding to each of the eight 3Y viral gene segments are KM922673–KM922680. 2.3. Adaptation of H7N7 influenza virus in mice Two mouse-adapted variants of the 3Y virus were derived from two independent series of sequential lungto-lung passages of virus in mice as described previously (Brown, 1990; Yu et al., 2014b). Briefly, female 3–5-weekold BALB/c mice (Merial-Vital Laboratory Animal Technology Co., Ltd., Beijing, China) were inoculated intranasally with 50 ml of allantoic fluid containing the wild type (WT) 3Y H7N7 virus under light isoflurane anesthesia. Lungs were harvested and homogenized 48 h after infection. The disrupted lung tissue was centrifuged to remove debris and 50 ml of the supernatant was used to inoculate the next naı¨ve mouse in the series. Virus was passaged mouseto-mouse seven times in two independent lines. After the seventh passage in each series of mice, viruses present in the final lung homogenates were cloned once by plaque purification in MDCK cells as described previously (Song et al., 2009; Yu et al., 2014b). The cloned viruses were independently amplified in the allantoic cavities of 10day-old chicken eggs for 48 h at 37 8C to prepare virus stocks. Two plaque-purified mouse-adapted variants of the original 3Y virus were obtained for further characterization and named MA-H1P7 and MA-H2P7. 2.4. Virus titration in eggs and MDCK cells Virus titers in virus stocks and homogenized organ samples were determined by end-point titration in eggs and/or MDCK cells as described previously (Yu et al., 2014b). Briefly, 10-fold serial dilutions of each sample were inoculated into eggs or onto MDCK cell monolayers grown in a 96 well culture plate. One hour after inoculation, the MDCK cell monolayer was washed with phosphate-buffered saline (PBS), and cultured in 100 ml of DMEM supplemented with 100 IU/ml penicillin, 100 mg/ ml streptomycin, 2 mM glutamine, and 2 mg/ml TPCKtreated trypsin. Forty-eight hours or seventy-two hours after inoculation, fluid from the allantoic cavity or supernatants of infected cell cultures were tested for the ability


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to agglutinate chicken erythrocytes as an indicator of virus replication. Infectious virus titers were reported as log10 EID50/ml(g) or log10 TCID50/ml, and were calculated from three replicates by the method of Reed–Muench (Reed and Muench, 1938).

the manufacturer’s protocol. Viral gene segments were sequenced by the Beijing Genomics Institute (Beijing, China). DNA sequences were analyzed and compared to the parental 3Y virus using the Lasergene sequence analysis software package (DNASTAR, Madison, WI).

2.5. In vivo experiments

2.8. Statistical analysis

The 50% mouse lethal dose (MLD50) of the parental 3Y virus and the two mouse-adapted isolates was measured using groups of three female 4–5-week-old BALB/c mice (Merial-Vital Laboratory Animal Technology Co., Ltd., Beijing, China) as described previously (Yu et al., 2014b). Briefly, mice were intranasally inoculated with 50 ml of 10fold serial dilutions of each indicated influenza virus in PBS under isoflurane sedation. Animals that showed signs of severe disease and weight loss >25% of their initial body weight were considered moribund and were humanely sacrificed. MLD50 values were calculated by the Reed– Muench method (Reed and Muench, 1938) after a 14-day observation period and expressed as EID50. Mice were additionally intranasally inoculated with 104 EID50 of the indicated viruses to measure the replicative capacity of mouse-adapted isolates as compared to the parental 3Y virus in the lungs. Three mice in each group were euthanized at 3 and 5 dpi, and the lungs were collected. In addition, to evaluate the tropism and replicative capacity of each virus in vivo, we euthanized three mice 3 days after inoculation with 106 EID50 the parental 3Y virus or mouse-adapted isolates and harvested the lungs, brains, intestines, livers, spleens, and kidneys. Organs were homogenized in 1 ml of PBS and viral titers in each of the organs were determined by titration in chicken eggs. Titers were calculated by the Reed–Muench method (Reed and Muench, 1938) and expressed as mean log10 EID50/g SD. The limit of virus detection was 0.75 log10 EID50/g. For calculation of the mean, samples with a virus titer of 6.5 log10 EID50, and the MLD50 of the MA-H1P7 and MA-H2P7 viruses were 3.5 log10 EID50 and 2.75 log10 EID50, respectively (Table 1). These results demonstrate that serial passage of WT 3Y resulted in viruses with markedly increased virulence in mice.

2.6. Analysis of replication kinetics in MDCK cells MDCK cells were infected with indicated influenza viruses at a multiplicity of infection (MOI) of 0.01 TCID50 (50% tissue culture infectious dose)/cell. After incubation, the cells were washed and overlaid with DMEM containing 2 mg/ml TPCK-treated trypsin. Supernatants were collected 12, 24, 36, 48, 60, and 72 h after infection and stored at 80 8C. Virus titer was determined by end-point titration in MDCK cells. Virus titers were expressed as mean log10 TCID50/ml SD. 2.7. RNA isolation, PCR amplification, and sequencing Viral RNA was isolated from the allantoic fluid of inoculated eggs using the RNeasy Mini kit (QIAGEN, Germantown, MD) according to the manufacturer’s instructions. Reverse transcription of viral RNA and subsequent PCR were performed using primers specific for each gene segment (sequences available upon request). PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Germantown, MD) according to

3.2. The mouse-adapted viruses replicate efficiently in MDCK cells We next evaluated the replicative ability of the WT 3Y and mouse-adapted H7N7 strains in MDCK cells (Fig. 2A). In vitro growth kinetics revealed that the MA-H1P7 and MA-H2P7 viruses grew faster and achieved higher titers than WT 3Y (Fig. 2A). MA-H2P7 grew to the highest titer and yielded approximately 7-fold more virus than WT 3Y in MDCK cells by 48 h post-inoculation (Fig. 2A). These data show that the enhanced virulence of the mouse-adapted H7N7 viruses correlates with increased viral replication in vitro. 3.3. The mouse-adapted H7N7 viruses display enhanced replication capacity and expanded tissue tropism in mice To determine if the mouse-adapted H7N7 strains replicate more efficiently than wild type virus in vivo, we inoculated mice with 104 EID50 of each virus and analyzed virus titers in the lungs on day 3 and 5 post-infection


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Fig. 1. Increased virulence of mouse-adapted H7N7 viruses in mice. Mice (n = 5) were inoculated intranasally with 50 ml containing 106 EID50 of mouse-adapted isolates (MA-H1P7 and MA-H2P7) or the parental wildtype H7N7 virus (WT) or mock inoculated (Mock; n = 5), and animals that lost more than 25% of their pre-infection weight were euthanized. (A) Morbidity was examined by recording the body weights of inoculated mice daily, and it is represented as a percentage of the weight on the day of inoculation (day 0). The average of each group is shown. The proportion of surviving mice in each group is indicated. (B) Mouse mortality after inoculation with 50 ml containing 106 EID50 of mouse-adapted isolates (MA-H1P7 and MA-H2P7) or the parental wild-type H7N7 virus (WT) or diluent (Mock). Table 1 MLD50 of wild-type 3Y H7N7 virus and mouseadapted H7N7 variants in BALB/c mice. Virusa WT 3Y MA-H1P7 MA-H2P7

MLD50 (log10 EID50) >6.5 3.5 2.75

a WT 3Y, A/Lesser White-fronted Goose/HuNan/ 412/2010 (H7N7); MA-H1P7, mouse-adapted 3Y; MA-H2P7, mouse-adapted 3Y.

(Fig. 2B). Both mouse-adapted variant viruses achieved higher lung viral titers on day 3 and day 5 post-infection. The titers of WT 3Y were 5.3 1.2 log10 EID50/g on day 3 postinfection and 6.3 0.9 log10 EID50/g on day 5 post-infection (Fig. 2B). In contrast, lung viral titers of MA-H1P7 and MAH2P7 were 7.9 0.9 log10 EID50/g and 8.4 0.5 log10 EID50/g, respectively, on day 3 post-infection, and 8.2 0.7 log10 EID50/ g and 7.1 0.1 log10 EID50/g on day 5 post-infection (Fig. 2B). These data show that the enhanced virulence of the mouseadapted H7N7 viruses correlates with increased viral replication in vivo. We then asked whether the increased virulence of the mouse-adapted H7N7 strains was due to an expansion of the tropism of the adapted variants. Mice were inoculated

Fig. 2. Growth characteristics of mouse-adapted H7N7 viruses in vitro and vivo. (A) MDCK cells were inoculated at a multiplicity of infection of 0.01 TCID50/cell with the parental wild-type H7N7 virus (WT) or a mouseadapted virus (MA-H1P7 or MA-H2P7). Supernatants were collected at the indicated time points and titrated in MDCK cells by TCID50. The average of three experimental replicates is shown with standard deviation indicated using error bars. (B) Mice (n = 3) were inoculated intranasally with 104 EID50 of the parental wild-type H7N7 virus (WT) or a mouse-adapted virus (MA-H1P7 or MA-H2P7). Viral loads in the lungs were determined at 3 and 5 days post-infection in eggs by EID50. Results are expressed as log10 EID50/g of tissue. The dotted line indicates the limit of detection. (C) Mice (n = 3) were infected with 106 EID50 of the parental wild-type H7N7 virus (WT) or a mouse-adapted virus (MA-H1P7 or MAH2P7). On day 3 post-infection, viral loads in the lungs, brains, intestines, livers, spleens, and kidneys were titrated in eggs by EID50. Results are expressed as log10 EID50/g of tissue. The dotted indicates the lower limit of detection of infectious virus. In each panel, the average of three replicates is shown with error bars indicating the standard deviation. *,8p < 0.05, when comparing MA-H1P7 and MA-H2P7 with WT respectively, as determined by one-way ANOVA. **,88p < 0.01, when comparing MA-H1P7 and MA-H2P7 with WT respectively, as determined by one-way ANOVA.

intranasally with 106 EID50 WT 3Y, MA-H1P7, or MA-H2P7. On day 3 post-infection, the lungs, brains, intestines, livers, spleens, and kidneys were collected and the viral load in each tissue was determined (Fig. 2C). Virus was recovered from the lungs of mice inoculated with WT 3Y, but was not detected in any other organ tested (Fig. 2C). In contrast, virus from mice inoculated with either mouse-adapted


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Table 2 Nucleotide mutations and amino acid substitutions identified in mouse-adapted H7N7 avian influenza viruses. Mouse-adapted virus MA-H1P7

Segment

Nucleotide position

Nucleotide mutation

Amino acid position

Amino acid substitution

PB2 PB1 PA

1879 353 513 1648 640

G!A G!T C!A C!A G!A

627 118 171 550 214

E!K R!I –a L!M G!R

1879 353 513 1024 1648 640 1115

G!A G!T C!A C!T C!A G!A G!A

627 118 171 342 550 214 372

E!K R!I – – L!M G!R S!N

HA MA-H2P7

PB2 PB1 PA

HA NA a

Synonymous mutation.

Table 3 Identification of H7N7 isolates with amino acid substitutions identified in mouse-adapted H7N7 variants. Amino acid position

Amino acid

Virus isolate

Accession no.

PB2

627

K

A/Netherlands/219/03(H7N7) A/Netherlands/219/2003(H7N7)

AY342413 GU053128

PB1 PA HA NA

118 550 214 372

I M R N

– – – –

– – – –

Segment

H7N7 strains was recovered from the lungs at higher titers when compared to mice inoculated with WT 3Y, and was also recovered from the brains, livers, spleens, kidneys, and intestines of mice on day 3 post-infection (Fig. 2C). These data demonstrate that mouse-adapted H7N7 strains replicate to higher titers in respiratory tissues and acquired the ability to replicate to detectable levels in the brain, liver, spleen, kidney, and intestines. 3.4. Mouse-adapted virus sequence analysis We sequenced the genomes of the MA-H1P7 and MAH2P7 viruses to identify the adaptive mutations responsible for the increased virulence in mice (Table 2). While sequence analysis revealed a number of synonymous and nonsynonymous mutations in the mouse adapted viruses, we focused on mutations that encoded amino acid substitutions for subsequent analyses. The MA-H1P7 and MA-H2P7 viruses both had an E!K substitution at PB2 position 627, an R!I substitution at PB1 position 118, an L!M substitution at PA position 550, and a G!R substitution at HA position 214 (Table 2). The MA-H2P7 virus also had an S!N substitution at NA position 372 that was not present in the MA-H1P7 virus (Table 2). To investigate whether the amino acid changes observed in mouse-adapted H7N7 strains generated in this study have been identified in other H7N7 isolates, we queried H7N7 sequences deposited in the GenBank database. We identified two isolates possessing a K residue at PB2 position 627, and found no evidence of H7N7 isolates possessing an I residue at PB1 position 118, an M residue at PA position 550, an R residue at HA position 214, or an N residue at NA position 372 (Table 3). It is worth

noting that the PB2 E627K substitution is known to play important roles in the adaptation of avian H5N1 viruses to mammalian hosts, and is associated with increased replicative capacity and pathogenesis in humans and mice (Gabriel et al., 2013; Hatta et al., 2001). 4. Discussion The potential pandemic threat of AIVs warrants research aimed at understanding the adaptive processes that confer enhanced AIV replication and virulence in mammals. Here, we use mice as a model for studying the mammalian adaptation of an H7N7 avian influenza virus isolated from wild waterfowl, and show that highly pathogenic variants can quickly emerge from a parental virus during limited serial passage in mice. Adapted viral variants displayed expanded tissue tropism and increased replication kinetics in vitro and in vivo when compared to the parental virus. Analysis of the variant virus genomes revealed the presence of multiple amino acid substitutions involved in the adaptation of the H7N7 avian influenza virus in mice. A number of amino acid substitutions have been implicated in the adaptation of avian influenza virus in mammals. For example, the PB2-E627K, PB2-D701N, and PB2-S714R substitutions have been consistently found to contribute to the adaptation of H5N1 avian influenza viruses in mammals (Czudai-Matwich et al., 2014; Hatta et al., 2001). The PB2-D701N, HA-G218W, and HA-T156N substitution were the major adaptive genetic determinants for increased growth and virulence of human H3N2 influenza virus in mice (Ping et al., 2010, 2011), and the majority of single and double NS1 mutations acquired


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Table 4 Identification of other subtype mouse-adapted viruses with amino acid substitutions identified in this study. Amino acid position

Mutation

Subtype

Reference

PB2

627

E!K

H3N2 H5N1 H6N1 H6N6 H7N1 H9N2

Ping et al. (2010) Min et al. (2013) Yu et al. (2014b) Tan et al. (2014) Yu et al. (2014c) Wang et al. (2012)

PB1 PA HA NA

118 550 214 372

R!I L!M G!R S!N

– – – –

– – – –

Segment

upon human H3N2 influenza virus adaptation to the mouse possessed properties of increased virulence (Forbes et al., 2012). While the PB2 E627K substitution has been reported to confer increased virulence in mammals in the context of other avian influenza subtypes, it has not been experimentally investigated for H7N7 subtype avian influenza viruses. In this report, we provide data to support a role for the PB2-E627K substitution in the adaptation and virulent phenotype of H7N7 mouseadapted viruses in mice. We also identified amino acid substitutions in the PB1, PA, and HA segment of mouseadapted variants that may contribute to the enhancement of virulence of H7N7 AIV in mammals. Notably, with the exception of PB2 627K, none of the adaptive changes identified in this study have been identified in other H7N7 isolates (Table 3). The investigation of the identified mutations was expanded to mutations identified in other mouse-adapted experiments with other subtypes. The PB2-E627K substitution also be found in the H3N2, H5N1, H6N1, H6N6, H7N1, and H9N2 subtype mouse-adapted influenza viruses (Table 4), indicating that PB2-E627K is a conserved mutation associated with these subtype influenza viruses adaptation to mice. In contrast, the other mutations (PB1-R118I, PAL550M, HA-G214R, and NA-S372N), which were identified in this study, were not found in the other subtype mouseadapted influenza viruses (Table 4), indicating that these mutations are a specific selection of H7N7 subtype. In conclusion, we found that increased virulence of mouse-adapted H7N7 AIVs was associated with accelerated viral growth in mammalian cells and expanded tissue tropism in vivo and some amino acid substitutions occurred in multiple gene segments of the mouse-adapted H7N7 AIVs. Additional studies will be required to isolate the role of each of these adaptive changes on viral virulence in mice. Extension of our findings to other animal models, including ferrets, pigs, and primates, will promote a better understanding of the molecular processes by which AIVs adapt to mammalian hosts. Finally, the identification of amino acid substitutions associated with mammalian adaptation of H7N7 viruses can be applied to ongoing surveillance activities which aim to assess the pandemic potential of circulating H7N7 AIVs. Conflict of interest There are no potential conflicts of interest.

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Multiple amino acid substitutions involved in the adaptation of H6N1 avian influenza virus in mice.

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