Influenza A viruses are important pathogens that infect both avian and mammalian species although these viruses are believed to have originated from avian influenza viruses (1
). Sixteen hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A viruses have been isolated from birds (1
). Predicting which of the subtypes of avian influenza viruses might be able to cross the species barrier and infect humans has proved difficult. This was demonstrated in 2013 by the unexpected emergence in humans of a low-pathogenic avian influenza (LPAI) H7N9 virus in China, which has caused over 400 confirmed human infections, with a fatality rate of about 30%, according to the WHO (http://www.who.int/influenza/human_animal_interface/influenza_h7n9/Risk_Assessment/en/
The H6 subtype of influenza viruses has the potential to cross the species barrier. It was first isolated from a turkey in 1965 and has been detected worldwide since then (4–7
). Influenza virus surveillance indicated that H6 viruses had become enzootic in the domestic ducks of southern China, mostly as H6N6 viruses (8
). Approximately one-third of H6 viruses isolated from live poultry markets in southern China could bind to human-type receptors, many could replicate in mice, and some could transmit efficiently among guinea pigs (9
). Serological surveys conducted in China and America indicated that H6 influenza viruses might have previously infected swine and humans (10
), and one-third of human volunteers inoculated with H6N1 or H6N2 viruses showed mild clinical symptoms with virus shedding (12
). H6N1 viruses caused significant morbidity and mortality in mice without prior adaptation (13
), and an H6N5 virus was lethal to mice and could be transmitted between ferrets by direct contact (14
In 2011, an H6N6 virus, A/swine/Guangdong/K6/2010 (GDK6), was isolated from a swine farm in southern China, and 3.4% of the pigs in the swine farms of this region were seropositive for H6 viruses (15
). The GDK6 virus belongs to the group II lineage of H6 viruses, which are the predominant H6 viruses in domestic ducks of southern China (8
). Subsequently, another H6N6 virus was isolated from swine in eastern China (16
), and the first known natural case of human infection with an avian H6N1 influenza virus was reported in Taiwan in 2013 (17
Adaptation is considered a primary impetus in evolution, and the process of natural selection in influenza A viruses appears to be mimicked by experimental adaptation in mice (18
). Although some avian influenza viruses can replicate efficiently in mice without prior adaptation, they are mostly avirulent, and adaptation of a virus in mice can lead to the emergence of mutations causing higher pathogenicity (18–21
). However, there is still limited knowledge of the molecular basis for the virulence of H6 viruses in mammals (20
Here, we focus on the molecular basis of pathogenicity in a mouse-adapted (MA) variant of the GDK6 (GDK6-MA) H6N6 virus. Using reverse genetics to generate reassortants of the wild type and a mouse-adapted virus, we identified the genes and mutations responsible for its higher pathogenicity in mice. A novel combination of substitutions in HA (H156N and S263R) and PA (I38M) led to the enhanced virulence of this H6N6 influenza A virus in mice.
MATERIALS AND METHODS
All animal studies were conducted under the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The animal experiments in our study were approved by the animal experimental ethics committee of the South China Agricultural University (approval number 2013-07).
Cells and viruses.
Adenocarcinomic human alveolar basal epithelial cells (A549), human embryonic kidney cells (293T), and Madin-Darby canine kidney (MDCK) cells were obtained from the Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences, China. Cells were propagated in growth medium containing Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel) and 1% Glutamax (Gibco, USA) at 37°C and 5% CO2 until they reached ∼80% confluence.
A/swine/Guangdong/K6/2010 (H6N6) (GDK6) was isolated as described previously (15
). Virus stocks were propagated in the allantoic cavities of 10-day-old specific-pathogen-free (SPF) embryonated hens' eggs (Merial, Beijing, China) at 37°C for 48 h. Allantoic fluid containing virus was harvested, aliquoted, and frozen at −80°C until used in experiments. Virus titers were measured by plaque assays in MDCK cells, and results were expressed as PFU counts (23
). All experiments with live viruses were performed in an enhanced animal biosafety level 3 (ABSL-3+) facility at the South China Agricultural University, Guangzhou, China.
Adaptation of the GDK6 virus in mice.
A mouse-adapted variant was derived from a series of sequential lung-to-lung passages in mice. Five 6-week-old female BALB/c mice (Guangdong Medical Laboratory Animal Center, China) were lightly anesthetized with dry ice, and 50 μl of allantoic fluid containing 106 PFU of wild-type GDK6 virus was inoculated intranasally (i.n.). At 3 days postinoculation (p.i.), three inoculated mice were euthanized, and the lungs were harvested and homogenized in 2 ml of sterile cold phosphate-buffered saline (PBS). The homogenate was centrifuged at 6,000 × g for 5 min at 4°C and filtered through a 0.22-μm-pore-size cellulose acetate filter (Millipore, USA). Fifty microliters of the centrifuged homogenate was used as the inoculum for the next passage. After 12 passages, the virus in the lung homogenate was cloned three times by plaque purification in MDCK cells, and the cloned virus, designated GDK6-MA, was inoculated into 10-day-old SPF embryonated hens' eggs and held for 48 h at 37°C to prepare a virus stock.
Molecular cloning and sequencing of the viral genes.
Viral RNA was extracted from aliquots containing virus using a NucleoSpin RNA Virus kit (Macherey-Nagel, Germany). As described previously, with minor modification in restriction enzyme cutting sites (24
), specific primers for each gene segment were used to perform reverse transcription of viral RNA and subsequent PCR. Amplified reverse transcription-PCR (RT-PCR) products were purified using a Wizard SV Gel and PCR Clean-Up System (Promega, USA). To sequence the viruses, the PCR products were cloned into the pJET 1.2 blunt-end cloning vector (Thermo Scientific, USA). At least three clones per gene were sequenced (performed by Shanghai Life Technologies Biotechnology Co., Ltd.).
) was used to predict a structural model of the HA of GDK6 based on its amino acid sequence (http://zhanglab.ccmb.med.umich.edu/I-TASSER/
). The structural interpretation of the PA N-terminal domain utilized the structure of the PA of A/Victoria/3/1975 (PDB identification number 2W69). PyMol, version 126.96.36.199 (www.pymol.org
), was used for visualization.
Construction of plasmids and virus rescue.
All reassortant viruses and the GDK6 and GDK6-MA viruses were generated by plasmid-based reverse genetics with the gene segments of GDK6 and GDK6-MA viruses cloned into the plasmid vector pHW2000 and amplified (27
). Mutations in the HA gene were introduced by PCR-based site-directed mutagenesis with primer pairs containing point mutations and confirmed by sequencing.
A monolayer of 293T cells with approximately 90% confluence in six-well plates was transfected with a mix of PB2, PB1, PA, HA, NP, NA, M, and NS plasmids (wild type or mutated; 0.25 μg of plasmid) using Lipofectamine LTX reagent (Invitrogen, USA) according to the manufacturer's instructions. Specifically, 0.25 μg of each plasmid was mixed and incubated with 6 μl of Lipofectamine LTX and 2 μl of Plus Reagent in 300 μl of Opti-MEM (Gibco, USA) for 15 min at room temperature and then added to the cells. After incubation for 1 h at 37°C with 5% CO2, 1 ml of Opti-MEM containing 1 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich, USA) and 0.2% bovine serum albumin (BSA; Sigma-Aldrich, USA) was added to each well. The supernatant of the 293T culture was harvested after a 24-h incubation, and 300 μl of supernatant was inoculated into 10-day-old SPF hens' eggs and held for 48 h at 37°C to prepare virus stock. The parental and recombinant viruses were plaque purified and confirmed by a hemagglutination inhibition (HI) test and sequencing. The virus fluid was stored at −80°C until used.
Mouse pathogenicity experiments.
Groups of eight 6-week-old female BALB/c mice (Guangdong Medical Laboratory Animal Center, China) were lightly anesthetized with dry ice and i.n. inoculated with 50 μl of allantoic fluid containing 106.4 PFU of virus or 50 μl of sterile PBS (mock group). At 3 days p.i., four mice were euthanized, three for virus titration of the organs and one for histology and immunohistochemistry. Virus was titrated in lungs, brains, kidneys, thymuses, spleens, and livers by plaque assays in MDCK cells. The remaining four mice in each group were monitored for weight loss and clinical signs for 12 days. The percent changes in body weights were calculated relative to the weight on day 0. Mice losing more than 25% of their body weight were humanely euthanized.
To exclude the possibility that unwanted mutations might have arisen in the PB2 segment within the mice during the single round of infection, the lungs of dead mice infected with viruses carrying residue 627E in PB2 [PB2(627E)] were collected, and the PB2 genes of these viruses were confirmed by sequencing. To determine the 50% mouse lethal dose (MLD50), groups of four 6-week-old female BALB/c mice were intranasally inoculated with 50 μl of 10-fold serial dilutions containing 103.4 to 106.4 PFU of virus in sterile PBS and observed for signs of morbidity over 12 days. The MLD50 value was calculated by the method of Reed and Muench and expressed as log10PFU.
For histopathological examinations, the lungs collected from mice at 3 days p.i. were immersed into 10% phosphate-buffered formalin, embedded in paraffin, and then cut into 5-μm-thick sections and stained with hematoxylin and eosin (H&E). The organs were examined microscopically. Additional sections were processed for immunohistological staining with rabbit polyclonal antibody against GDK6 virus (prepared in our laboratory). Goat anti-rabbit IgG-horseradish peroxidase (HRP; SunShineBio, China) directed against the primary antibody was used as a secondary antibody. Specific antigen-antibody reactions were visualized by means of 3,3′-diaminobenzidine tetrahydrochloride.
Growth dynamics of viruses.
The parental and recombinant viruses were inoculated into MDCK cell monolayers (multiplicity of infection [MOI] of 0.01 PFU) or A549 cell monolayers (MOI of 0.1 PFU) with DMEM containing 0.5% BSA and 1 μg/ml TPCK-treated trypsin and incubated at 37°C with 5% CO2. Cell supernatants were harvested every 12 h until 60 h postinoculation (hpi).
Single-replication-cycle experiments were performed to confirm the early-stage viral replication. The parental and recombinant viruses were inoculated into MDCK cell monolayers or A549 cell monolayers (MOI of 5 PFU) with DMEM containing 0.5% BSA and 1 μg/ml TPCK-treated trypsin and incubated at 37°C with 5% CO2. Cell supernatants were harvested at 6 hpi.
Virus in supernatants was titrated for infectivity in MDCK cells by plaque assays.
Minigenome assay for polymerase activity.
293T cells were transfected with 0.25 μg of pHW2000-PB2 (wild type or mutated), pHW2000-PB1, pHW2000-PA (wild type or mutated), pHW2000-NP, the luciferase reporter plasmid pPolI-Luci-T, and an internal control plasmid expressing Renilla luciferase (Promega, USA) using Lipofectamine LTX reagent (Invitrogen, USA) as recommended by the manufacturer. After 48 h of transfection, cell extracts were harvested and lysed with a Dual-Luciferase Reporter Assay System (Promega, USA), and luciferase activity was measured using a Modulus Single Tube reader (Turner Biosystems, USA).
The swine H6N6 viruses from China were the first known H6 subtype viruses to infect mammals (15
). The HA and NA of the virus isolated from Guangdong (GDK6) differed from those in the majority of H6 viruses. Substitutions A138S and G228S at highly conserved positions in the receptor binding region of GD6K HA would be expected to favor binding of α2,6-sialic acids (SAs) (31–33
). The 11-amino-acid deletion (positions 59 to 69) in the NA stalk region should affect NA activity (34
). These factors would alter the balance between the HA and NA (35
) and may have facilitated the infection by GDK6 in swine.
As seen in other swine or avian H6N6 viruses (9
), the GDK6 virus was almost avirulent in mice although it replicated efficiently in their lungs. Whether and, if so, how it could adapt to become pathogenic in mice and potentially other mammals was not known. After multiple serial passages of GDK6 virus in mouse lungs, a virulent mouse-adapted virus GDK6-MA was generated. This virus differed from GDK6 by 5 amino acid substitutions in three gene segments (PB2 E627K, PA I38M, and HA L111F, H156N, and S263R) and replicated to higher titers than GDK6 in the lungs of infected mice.
Consistent with earlier studies (29
), a recombinant GDK6 virus containing PB2(E627K), rGDK6-MA (PB2), had enhanced pathogenicity and replication in mice. Polymerase activity increased when PB2(627K) was present. While recombinant GDK6 viruses containing either the mouse-adapted HA or PA segment alone were not as virulent, the virus containing both segments had similar virulence to rGDK6-MA. Further investigation of the HA substitutions in the mouse-adapted virus showed that PA(I38M) and HA(H156N S263R) were responsible for the increased virulence in mice.
Methionine at position 38 of PA is rare in influenza A viruses, with no H6 and only seven other viruses having this residue. The N-terminal domain of PA (positions 1 to 256) is involved in protein stability, endonuclease activity, cap binding, promoter binding, and induction of the nuclear accumulation of PB1 (37–39
). The PA(I38M) mutation increased transcription activity by the viral polymerase in 293T cells in vitro
but did not influence the replication kinetics in MDCK or A549 cells.
The HA H156N and S263R substitutions that contribute to virulence are located on the globular head of HA1. H156N results in a potential N-linked glycosylation site not seen in other H6 viruses. Glycosylation in this region of HA can reduce receptor avidity (40
) or reduce dependence on the NA for release from the receptor (41
). This may be beneficial for HA-NA balance in the presence of a stalk deletion in the NA (41
), which occurs in the NA of GDK6. H156N in HA, or the potentially additional N-linked glycoside, significantly enhanced the cytopathic effect on MDCK cells and increased early-stage viral replication in MDCK cells, probably reflecting a contribution to the HA-NA balance of the virus in mammalian systems. HA(S263R) did not enhance viral replication either in vivo
or in vitro
, and the mechanism by which it contributes to the enhanced virulence of rGDK6-MA (HA, PA) is unknown.
The viral titers in the lungs of mice infected with the rGDK6-MA (HA, PA) or rGDK6-MA (PB2) were similar to the titer in rGDK6-infected mice at 3 days p.i. This suggests that the increased virulence of the mouse-adapted virus did not directly result from the viral titer in the lungs, as has been reported previously (20
The adaptation and pathogenicity of an influenza virus to a new host are clearly polygenic effects, as demonstrated here and in other studies (22
). Here, we found that while the individual mouse-adapted HA or PA segment only moderately enhanced the virulence of the GDK6 virus, the combination of these segments led to a virulent virus. We have identified key amino acids in the PA and HA proteins that significantly enhance viral pathogenicity in mice and can compensate in mammals for the lack of 627K in PB2. Our study suggests that multiple strategies can be utilized by influenza viruses for efficient adaptation in mammals, and it has identified a novel virulence determinant for mammalian influenza viruses.
We acknowledge scientific support from George Fu Gao, Institute of Microbiology, Chinese Academy of Sciences, and the colleagues of Nanchang Center for Disease Control and Prevention. We thank Yi Guan, University of Hong Kong, for his helpful critical review and revision of the manuscript.
This work was supported by the National Key Basic Research Program (Project 973) of China (grant no. 2011CB504700-G), the Science and Technology Projects of Guangdong province (2012B020306005), the International Science and Technology Cooperation Program (2010DFB33920), and the Modern Agricultural Industry Technology System (CARS-36).
The funding organizations had no role in the study design, data collection and analysis, ownership of the materials, or preparation of the manuscript.
We declare that we have no conflicts of interest.