Arch Virol (2015) 160:917–927 DOI 10.1007/s00705-015-2337-y
ORIGINAL ARTICLE
Comparison of biological characteristics of H9N2 avian influenza viruses isolated from different hosts Yinbiao Zhu • Yang Yang • Wei Liu • Xin Liu • Da Yang • Zhihao Sun • Yong Ju Sujuan Chen • Daxin Peng • Xiufan Liu
•
Received: 9 September 2014 / Accepted: 9 January 2015 / Published online: 24 January 2015 Ó Springer-Verlag Wien 2015
Abstract The pathogenicity and transmissibility of H9N2 influenza viruses has been widely investigated; however, few studies comparing the biological characteristics of H9N2 viruses isolated from different hosts have been performed. In this study, eight H9N2 viruses, isolated from chickens (Ck/F98, Ck/AH and Ck/TX), pigeons (Pg/XZ), quail/(Ql/A39), ducks (Dk/Y33) and swine (Sw/YZ and Sw/TZ) were selected, and their biological characteristics were determined. The results showed that all H9N2 viruses maintained a preference for both the avian- and humantype receptors, except for Sw/TZ, which had exclusive preference for the human-type receptor. The viruses replicated well in DF-1 and MDCK cells, whereas only three
isolates, Ck/F98, Ck/TX and Sw/TZ, could replicate in A549 cells and also replicated in mouse lungs, resulting in body weight loss in mice. All H9N2 viruses were nonpathogenic to chickens and were detected in the trachea and lung tissues. The viruses were shed primarily by the oropharynx and were transmitted efficiently to naı¨ve contact chickens. Our findings suggest that all H9N2 viruses from different hosts exhibit efficient replication and contact-transmission among chickens, and chickens serve as a good reservoir for the persistence and interspecies transmission of H9N2 influenza viruses.
Introduction Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2337-y) contains supplementary material, which is available to authorized users. Y. Zhu Y. Yang X. Liu D. Yang Z. Sun Y. Ju S. Chen D. Peng (&) X. Liu College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, People’s Republic of China e-mail:
[email protected];
[email protected] Y. Zhu Y. Yang X. Liu D. Yang Z. Sun Y. Ju S. Chen D. Peng X. Liu Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, People’s Republic of China Y. Zhu Y. Yang X. Liu D. Yang Z. Sun Y. Ju S. Chen D. Peng X. Liu Jiangsu Research Centre of Engineering and Technology for Prevention and Control of Poultry Disease, Yangzhou 225009, People’s Republic of China W. Liu China Animal Disease Control Center, Beijing 100123, People’s Republic of China
H9N2 avian influenza viruses (AIVs) are prevalent in Asia and can cause mild to severe disease in multiple poultry species [1, 12, 27]. In China, the H9N2 virus was first isolated from chickens in Guangdong province in 1994, and since then, H9N2 viruses have been well established in land-based poultry, resulting in large economic losses to the poultry industry [18, 21]. G1-, Y280- and BJ-94-, represented by the prototype viruses A/quail/Hong Kong/ G1/97, A/duck/Hong Kong/Y280/97, and A/chicken/Beijing/1/1994, respectively, were three distinct major lineages in domestic poultry in the mid-1990s [12]. Since 2000, the previously predominant BJ-94 lineage has been gradually replaced by the F98-like virus, represented by A/chicken/Shanghai/F/98, in both southern and northern China. Additionally, multiple genotypes of the H9N2 virus were generated through complicated reassortment of these different H9N2 lineages [34, 51]. In addition to replicating in poultry, H9N2 AIVs occasionally expand their host ranges to infect mammalian
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species, including pigs [8] and humans, causing mild disease [6, 30]. Recently, a considerable proportion of sera positive for H9 influenza virus was found in the poultryexposed human population [47, 50], suggesting the frequent occurrence of mild human infections. Further evidence for an expanded mammalian host range is the increase in the number of H9N2 isolates with a leucine at position 226 (L226, H3 numbering) in the receptor-binding site (RBS) of the haemagglutinin (HA) protein. L226 is a critical motif for enhanced viral binding affinity for a2,6linked sialic acid (SAa2,6) receptors and efficient replication in mammalian hosts [19, 43, 44]. During our surveillance for AIVs in eastern China, a novel triple reassortant genotype of the H9N2 virus that has the backbone of an F98-like virus with PB2 and M gene segments from a G1-like virus and an HA gene segment derived from a Y280-like virus emerged in chickens [19]. Moreover, this novel H9N2 AIV genotype was also isolated from other species. There are many studies on the pathogenicity and transmissibility of H9N2 viruses [12, 18, 29, 45]. However, few studies are available regarding the biological characterisation of H9N2 viruses from different hosts. In this study, we selected a panel of eight H9N2 viruses from different hosts to evaluate their biological characteristics, including their receptor-binding ability, their replication efficiency in chicken-origin fibroblasts (DF-1), Madin-Darby canine kidney (MDCK) cells, and human type II alveolar epithelial (A549) cells, and their pathogenicity in chickens and mice.
Materials and methods Ethics statement The Jiangsu Administrative Committee for Laboratory Animals (Permit number SYXKSU-2007-0005) approved all animal studies according to the guidelines of Jiangsu Laboratory Animal Welfare and Ethical of Jiangsu Administrative Committee of Laboratory Animals. Viruses Eight H9N2 viruses were used in the present study (Table 1, Table S1) and were isolated from different hosts, including chickens, ducks, quail, pigeons and swine, in our laboratory during the epidemiological surveillance for AIVs in eastern China. The sequences of the viruses A/chicken/Shanghai/F/98 [23], A/chicken/Taixing/10/ 2010, A/pigeon/Xuzhou/1/2011, A/quail/Wuxi/7/2010 and A/duck/Wuxi/7/2010 [19] have been described previously, and the sequences of the viruses A/swine/Taizhou/5/2008, A/swine/Yangzhou/1/2008 and A/chicken/Anhui/1/2008
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were available in GenBank under accession numbers HM998911-HM998918, HM998919-HM998926, and KP081530-KP081537, respectively. All viruses were grown in the allantoic cavities of 10-day-old embryonated chicken eggs at 37 °C for 72 h. Stock viruses were aliquoted and stored at -70 °C until use. The viral titres were determined either by inoculating 100 lL of 10-fold dilutions of virus into 10-day-old embryonated chicken eggs for the 50 % egg infectious dose (EID50) or in cell culture for the 50 % tissue culture infectious dose (TCID50), as described by Reed and Muench [32]. Phylogenetic analysis Phylogenetic relationships of each gene of the viruses were determined by the neighbor-joining method using the software MEGA 5.05 (http://www.megasoftware.net/) [10, 37]. Cells DF-1, Madin-Darby canine kidney (MDCK), and human type II alveolar epithelial (A549) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % (v/v) foetal bovine serum (Hyclone, UT, USA) at 37 °C and 5 % CO2. Receptor-binding assay The receptor specificity of the viruses was first examined using a haemagglutination assay as described previously [35]. Briefly, a 100-lL aliquot of a 10 % (v/v) suspension of goose red blood cells (GRBCs) was treated with 1.25 units of a2,3-sialidase (Takara) for 1 h at 37 °C. The treated erythrocytes were washed twice and then adjusted to a final working concentration (0.5 %, v/v) with phosphate-buffered saline (PBS). Viruses in a volume of 50 lL were serially diluted using 50 lL PBS and were mixed with 50 lL 0.5 % of GRBCs in a 96-well microtitre plate at room temperature. The haemagglutination assay titres were read after a 15-minute incubation. The receptor-binding specificity was further analysed using a solid-phase direct binding assay as described previously [2]. Briefly, the synthetic sialylglycopolymers Neu5Aca2-3Galb1-4GlcNAcb (30 SLN)-PAA-biotin and Neu5Aca2-6Galb1-4GlcNAcb (60 SLN)-PAA-biotin (GlycoTech) were serially diluted with PBS and added to a 96-well microtitre plate coated with streptavidin (Pierce). The plates were blocked with 2 % skim milk powder in PBS, followed by the addition of 128 haemagglutination assay units of live virus per well. The monoclonal antibody 3B3B6 (available in our laboratory) against the HA protein
Chicken
Chicken
Pigeon
Quail
Duck
Swine
Swine
A/chicken/Anhui/1/2008
A/chicken/Taixing/10/2010
A/pigeon/Xuzhou/1/2011
A/quail/Wuxi/7/2010
A/duck/Wuxi/7/2010
A/swine/Yangzhou/1/2008
A/swine/Taizhou/5/2008
Sw/TZ
Sw/YZ
Dk/Y33
Ql/A39
Pg/XZ
Ck/TX
Ck/AH
Ck/F98
Abbreviation
F98 (95.9)
(95.1)
(95.5)
(86.9) F98
F98
F98 (95.8)
G1
F98 (94.5)
F98 (95.1)
F98 (94.7)
F98 (94.8)
G1
(95.2)
(86.8) (87.3)
F98
(95.6)
(86.9)c G1
F98
F98
PB1
G1
F98
PB2
Genetic sourcea
(97.1)
F98
(97.3)
F98
F98 (95.1)
(96.4)
F98
(95.5)
F98
(95.2)
F98
(97.0)
F98
F98
PA
(91.4)
Y280
(91.6)
Y280
Y280 (91.2)
(91.3)
Y280
(90.0)
Y280
(91.3)
Y280
(91.6)
Y280
F98
HA
(95.9)
F98
(95.5)
F98
F98 (95.2)
(95.2)
F98
(95.4)
F98
(95.3)
F98
(95.5)
F98
F98
NP
(93.4)
F98
(93.7)
F98
F98 (92.7)
(92.7)
F98
(92.5)
F98
(92.1)
F98
(93.8)
F98
F98
NA
(93.8)
G1
(94.2)
G1
G1 (92.8)
(93.0)
G1
(93.2)
G1
(93.5)
G1
(93.9)
G1
F98
M
(97.1)
F98
(97.2)
F98
F98 (95.9)
(96.5)
F98
(95.6)
F98
(95.6)
F98
(97.1)
F98
F98
NS
PSRSSR
PSRSSR
PSRSSR
PSRSSR
PSRSSR
PSRSSR
PSRSSR
PARSSR
Cleavage site in HA
L
L
L
L
L
L
L
Q
226
K
K
K
K
K
K
K
R
363
Residues in HAb
D
D
D
D
D
D
D
D
253
Q
Q
Q
Q
Q
K
Q
Q
591
E
E
E
E
E
E
E
E
627
Residues in PB2
L
L
L
L
L
L
L
L
672
Residue in PA
c
b
Residue at 226 in HA (H3 numbering) Nucleotide sequence identity (%) to the virus A/chicken/Shanghai/F/98
PB1, polymerase basic protein; PA, polymerase acidic protein; HA, haemagglutinin; NP, nucleocapsid protein; NA, neuraminidase; M, matrix; NS, nonstructural; Y280, Y280-like (represented by A/duck/Hong Kong/Y280/97); F98, F98-like (represented by A/Chicken/Shanghai/F/98); G1, G1-like (represented by A/Quail/Hong Kong/G1/97)
a
Chicken
A/chicken/Shanghai/F/98
Host
Table 1 H9N2 influenza viruses used in this study
Comparison of H9N2 AIVs 919
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920
of the H9N2 virus was diluted in PBS and added to each well. Bound antibody was detected by the sequential addition of HRP-conjugated rabbit anti-mouse IgG antibody and tetramethylbenzidine substrate solution. The reaction was stopped with 50 ll of 1 M H2SO4, and the optical density (OD) was measured at 450 nm. The experiment was performed in triplicate. Replication kinetics in cells DF-1, MDCK and A549 cells were infected with 0.01 50 % tissue culture infective dose (TCID50) of virus for 1 hour at 37 °C. The cells were then washed twice with PBS and incubated in the appropriate medium containing a suitable amount of N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma, St. Louis, MO, USA) at 37 °C. For DF-1 cells, only 0.5 lg/ml TPCK-trypsin was used because the cells are highly sensitive to TPCK-trypsin, whereas 2 lg/ml TPCK-trypsin was used for both the MDCK and A549 cells. The supernatants were harvested at 12, 24, 48 and 72 h post-inoculation (p.i.) and stored at -70 °C. The viral titres in these supernatants were titrated by TCID50 analysis in MDCK cells based on the method of Reed and Muench [32].
Y. Zhu et al.
Virulence and transmission in chickens Six-week-old specific-pathogen-free (SPF) White Leghorn chickens were divided into eight groups and inoculated intranasally with 106 EID50 of virus in a 0.2-ml volume (n = 8 per group). Five chickens for each group were housed with virus-inoculated chickens one day after inoculation as contact groups. All chickens were monitored daily for clinical signs for up to 14 days. Virus replication in the organs, including the trachea, lung, kidney, spleen and brain, was examined in three inoculated chickens in each group on the third day postinfection (dpi). Oropharyngeal and cloacal swabs were collected from chickens at 3, 5 and 7 dpi and resuspended in 1 ml PBS. All tissue and swab samples were titrated for virus infectivity by EID50 assay. Statistical analysis The viral titres in supernatants from H9N2-virus-infected DF-1, MDCK and A549 cell cultures were compared by analysis of variance (ANOVA). For the animal experiments, the data were analysed by two-way ANOVA with Student’s t-test. Differences were considered significant at P \ 0.05.
Thermostability Thermostability was examined according to a previous study [28] with minor modifications. The initial titres for the stock viruses in the allantoic fluid were determined by haemagglutination assay. Prior to the incubation, starting titres of the viruses were standardised to 256 haemagglutination assay units. Next, influenza stocks were divided into several aliquots and placed in a 56 °C water bath for intervals up to 180 minutes and quick-frozen for viral titre determination.
Results Phylogenetic analysis
Animal experiments
Nucleotide sequence analysis of the eight genes of the viruses revealed that seven of eight H9N2 viruses were triple reassortants with a backbone from an F98-like virus, an HA gene from a Y280-like virus, and an M gene from a G1-like virus, and the PB2 genes of four H9N2 viruses were from G1-like virus. Analysis of the deduced amino acid sequence showed that all of the viruses possessed
Pathogenicity in mice
Table 2 Haemagglutination activity of H9N2 influenza viruses
Eight groups (n = 8 per group) of 6-week-old female BALB/c mice were lightly anaesthetised with isoflurane and inoculated intranasally with 106 EID50 in 50 lL of diluted virus in sterile PBS. Mice inoculated with sterile PBS were used as negative controls. The mice were monitored for clinical signs of infection and weighed daily for 14 days. At 3 dpi, three mice from each group were killed, and the lungs were removed and homogenised in 1 mL PBS. The viral titres were determined by EID50 determination. A haemagglutinin inhibition (HI) test was conducted to confirm seroconversion of the mice at 21 dpi.
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Virus
HA titre (nlog2) Untreated GRBCs
Treated GRBSsa
Ck/F98
8
7
Ck/AH
8
4
Ck/TX
9
8
Pg/XZ
8
7
Ql/A39
8
5
Dk/Y33
7
6
Sw/YZ
8
6
Sw/TZ
9
9
a
The goose red blood cells were treated with a2,3-sialidase
Comparison of H9N2 AIVs
921
Fig. 1 Solid-phase receptor-binding assay for H9N2 influenza viruses. The direct binding property of viruses to sialylglycopolymers, including either 30 SLN-PAA or 60 SLN-PAA, was detected. The results represent the means ± SD of triplicate experiments
627E in the PB2 gene, a 3-amino-acid deletion in the NA gene, and 226L in the HA gene except for Ck/F98, which had 226Q in the HA gene (Table 1). Receptor-binding assay To determine the receptor-binding properties of these viruses, the H9N2 viruses were first examined using the haemagglutination assay. The result showed that all viruses reacted well with both the untreated and a2,3-sialidasetreated GRBCs (Table 2), indicating that they all have high affinity for a2,6-linked sialic acid on the surface of GRBCs. To further examine the receptor-binding specificity, particularly for a2,3-linked sialic acid, a solid-phase binding assay using a glycan complex was used. The Sw/
TZ virus bound only to 60 SLN, indicating a binding preference for a2,6-sialoglycan. The other viruses bound to both 30 SLN and 60 SLN, indicating a binding preference for both a2,3- and a2,6-sialoglycan. The virus Ck/F98 showed similar binding ability for both sialoglycans, and the other viruses showed enhanced binding to a2,6-sialoglycan (Fig. 1). Replication kinetics The replication kinetics of these eight viruses were studied in MDCK, DF-1 and A549 cells. In MDCK cells, all of the viruses replicated efficiently (Fig. 2a). The titres of the Dk/ Y33, Pg/XZ and Sw/YZ viruses were significantly lower than those of the CK/F98 virus, whereas the titres of the
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Fig. 2 Replication kinetics of H9N2 influenza viruses in MDCK (a), DF-1 (b) and A549 (c) cells. The cells were infected at a multiplicity of infection of 0.4 TCID50/cell. The virus titres at the indicated time postinoculation were titrated in MDCK cells and expressed as log10 TCID50/ml. Each point indicates the mean of three independent experiments. The statistical analysis was performed between the Ck/98 virus and other viruses. *P \ 0.05; **P \ 0.01; ***P \ 0.001
Only the Ck/F98, Ck/TX and Sw/TZ viruses replicated in A549 cells, and the Ck/TX and Sw/TZ viruses grew better than the CK/98 virus (Fig. 2c). Thermostability
Fig. 3 Thermostability of H9N2 viruses at 56 °C. The diluted viruses with identical titres were divided into several aliquots and incubated at 56 °C up for 120 minutes. The viral titres were determined at the indicated time points after incubation, and their values represent the means of two independent experiments
other viruses were similar to those of the CK/F98 virus. Similarly, all viruses also replicated well in DF-1 cells (Fig. 2b). The Pg/XZ and Sw/TZ viruses showed slow replication at 24 h postinfection compared with the CK/98 virus. There was no significant difference in viral titre among the viruses at the end of the observation period.
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To evaluate the thermostability of the viruses, the H9N2 viruses were incubated at 56 °C. The chicken-origin Ck/F98, Ck/TX and Ck/AH viruses exhibited excellent thermostability, as evidenced by only a slight decrease in haemagglutination titre from 8 log2 to 7 log2 during the entire incubation period (Fig. 3). By contrast, the quail-origin Ql/ A39 and duck-origin Dk/Y33 viruses were sensitive to treatment at 56 °C, with haemagglutination assay titres significantly decreasing from 8 log2 to zero after a 30-minute incubation. The remaining viruses were also thermostable. Pathogenicity in mice To investigate the potential pathogenicity of these H9N2 viruses to mammals, BALB/c mice were infected intranasally (i.n.) with H9N2 viruses at 106 EID50. Only three viruses, Ck/F98, Ck/TX and Sw/TZ, induced slight weight loss compared to the PBS-treated mice during the observation period. The mice lost less than 10 % of their initial weight by 3 dpi and were fully recovered as early as 5 dpi (Fig. 4a).
Comparison of H9N2 AIVs
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Fig. 4 Average body changes (a) and viral loads in the lungs (b) of BALB/c mice infected with H9N2 viruses. Six-week-old female BALB/c mice (n = 8/group) were inoculated intranasally with 50 lL containing 106 EID50 of the viruses or PBS (mock). Each group was monitored for 14 days, and the results represent the mean of the body
weight change. Three mice were randomly selected and euthanised at 3 dpi, and the viral loads in the lungs were expressed as the log10 EID50/ml. The dashed horizontal line indicates the lower limit of detection
Table 3 Titres of H9N2 influenza viruses in the organs of infected chickens
were shed from both the oropharynx and cloaca in the inoculated chickens within 7 dpi. Viral shedding detected in oropharyngeal swabs peaked at 3 dpi and decreased significantly at 5 dpi, and no viral shedding was detected at 7 dpi. At 3 dpi, the titres of the oropharyngeal swabs from CK/F98-infected chickens were significantly lower than those in the four groups of chickens infected with Ck/AH, Ck/TX, Sw/YZ and Sw/TZ. All viruses were also shed from cloaca, but the titres were near the limit of detection (1 log10EID50/0.1 ml) and were significantly lower than in the oropharyngeal swabs (Table 4). To investigate horizontal transmission of these viruses, five SPF chickens were housed with chicken challenged with viruses. During the observation period, the chickens in the contact group showed no clinical disease. Viral shedding in the contact chickens was similar to that in the inoculated chickens, with slight differences. Viral shedding in the oropharynx of the majority of contact chickens was prolonged and was detected at 7 dpi, except for the contact chickens with the Pg/XZ or Sw/YZ challenge group. Viral shedding from the cloaca in the contact chickens was not as efficient as in the inoculated chickens. At 5 dpi, only four viruses, Ck/F98, Ck/AH, Ck/TX, and Dk/Y33, were shed from the cloaca in the contact chickens. The contact chickens with the Sw/TZ challenge group showed no viral shedding at any time point post-contact (Table 4).
Virus
Titre (log10 EID50/g ± SD) Trachea
Lung
Spleen
Kidney
Brain
Ck/F98
3.93 ± 0.95
3.53 ± 0.92
NDa
ND
ND
Ck/AH
5.13 ± 0.21
3.10 ± 0.17
ND
ND
ND
Ck/TX Pg/XZ
4.93 ± 0.85 5.67 ± 0.40
3.43 ± 0.75 3.13 ± 0.71
ND ND
ND ND
ND ND
Ql/A39
4.40 ± 0.46
2.67 ± 0.21
ND
ND
ND
Dk/Y33
3.60 ± 0.30
2.50 ± 0.10
ND
ND
ND
Sw/YZ
5.60 ± 0.97
2.47 ± 0.06
ND
ND
ND
Sw/TZ
4.63 ± 1.10
2.67 ± 0.21
ND
ND
ND
a
Not detectable
The viral titres in the lungs of the challenged mice at 3 dpi were also determined. Of the eight H9N2 viruses, only three, Ck/TX, Ck/F98 and Sw/TZ, replicated in mouse lungs, with mean titres ranging from 2.68 to 3.06 log10EID50/0.1 ml (Fig. 4b). The mice in all groups seroconverted by 21 dpi based on the HI assay, with titres between 80 and 320. Pathogenicity and transmission in chickens To compare the pathogenicity of these eight viruses in chickens, we inoculated SPF chickens intranasally with 106 EID50 of virus in a volume of 200 lL. None of the experimentally infected chickens showed clinical disease within 14 dpi. At 3 dpi, all viruses replicated in the tracheas (3.60 to 5.67 log10EID50/g) and lungs (2.47 to 3.53 log10EID50/g) only, with no virus detected in the brains, spleens and kidneys (Table 3). Viral shedding was detected in the oropharyngeal and cloacal swabs from all groups at 3, 5 and 7 dpi. All viruses
Discussion Recently, increasing numbers of H9N2 AIV isolates have been found to possess the amino acid leucine at position 226 (by H3 numbering) in the HA glycoprotein. The L226 motif is associated with enhanced binding preference for the human-like receptor. Consistent with this finding, the
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Table 4 Viral shedding in oropharyngeal and cloacal swabs from infected chickens Shedding: log10 (EID50/0.1ml ±SDa) (No. of positive swabs/No. of total swabs)
Virus
3 dpi
Ck/F98
Infectedb Contact
Ck/AH
c
5 dpi
7 dpi
Oropharyngeal swabs
Cloacal swabs
Oropharyngeal swabs
Cloacal swabs
Oropharyngeal swabs
Cloacal swabs
3.79 ± 0.54 (5/5)
1.24 ± 0.37 (3/5)
2.13 ± 0.55 (5/5)
1.5 ± 0.23 (4/5)
NDd (0/5)
ND (0/5)
3.56 ± 0.10 (5/5)
1.15 ± 0.30 (3/5)
5.05 ± 0.58 (5/5)
1.22 ± 0.26 (4/5)
2.46 ± 0.07 (5/5)
ND (0/5)
Infected
5.96 ± 0.37 (5/5)
2.46 ± 0.07 (4/5)
1.75 ± 0.55 (5/5)
1.39 ± 0.23 (4/5)
ND (0/5)
ND (0/5)
Contact
5.73 ± 0.09 (5/5)
1.28 ± 0.28 (4/5)
3.56 ± 1.84 (5/5)
1.11 ± 0.23 (2/5)
1.20 ± 0.38 (4/5)
ND (0/5)
Ck/TX
Infected
6.68 ± 0.79 (5/5)
1.24 ± 0.12 (4/5)
1.46 ± 0.07 (5/5)
1.11 ± 0.24 (3/5)
ND (0/5)
ND (0/5)
Contact
6.28 ± 0.39 (5/5)
1.05 ± 0.10 (5/5)
1.68 ± 1.00 (5/5)
1.04 ± 0.11 (4/5)
1.19 ± 0.38 (2/5)
ND (0/5)
Pg/XZ
Infected
5.19 ± 0.61 (5/5)
1.46 ± 0.07 (4/5)
2.02 ± 0.90 (5/5)
1.12 ± 0.23 (3/5)
ND (0/5)
ND (0/5)
Contact
6.42 ± 0.07 (5/5)
1.29 ± 0.27 (4/5)
3.39 ± 0.77 (5/5)
ND (0/5)
ND (0/5)
ND (0/5)
Infected
4.94 ± 0.51 (5/5)
1.11 ± 0.23 (2/5)
2.61 ± 1.02 (5/5)
1.04 ± 0.11 (2/5)
ND (0/5)
ND (0/5)
Contact
5.79 ± 0.63 (5/5)
1.15 ± 0.31 (2/5)
4.85 ± 1.75 (5/5)
ND (0/5)
1.32 ± 0.52 (2/5)
ND (0/5) ND (0/5)
Ql/A39 Dk/Y33
Infected
4.57 ± 0.78 (5/5)
1.21 ± 0.27 (3/5)
2.89 ± 1.51 (5/5)
1.04 ± 0.11 (2/5)
ND (0/5)
Contact
2.19 ± 0.70 (5/5)
1.12 ± 0.23 (4/5)
3.90 ± 0.93 (5/5)
1.15 ± 0.31 (4/5)
2.83 ± 1.15 (5/5)
ND (0/5)
Sw/YZ
Infected
5.70 ± 0.45 (5/5)
1.26 ± 0.27 (4/5)
1.75 ± 0.55 (5/5)
1.04 ± 0.12 (3/5)
ND (0/5)
ND (0/5)
Sw/TZ
Contact Infected
6.11 ± 0.63 (5/5) 6.07 ± 0.68 (5/5)
1.26 ± 0.34 (4/5) 1.21 ± 0.27 (3/5)
1.41 ± 1.01 (4/5) 2.46 ± 0.07 (5/5)
ND (0/5) 1.15 ± 0.31 (2/5)
ND (0/5) ND (0/5)
ND (0/5) ND (0/5)
Contact
5.79 ± 0.51 (5/5)
ND (0/5)
2.24 ± 1.14 (5/5)
ND (0/5)
1.26 ± 0.49 (3/5)
ND (0/5)
a
For statistical purposes, a value of 1 was set as the lower limit of detection
b
Chickens were inoculated with the virus
c
Chickens were in contact with inoculated chickens
d
Not detectable
Ck/F98 virus with Q226 in the HA protein showed a similar binding preference for a2,3- and a2,6-sialoglycan. The other viruses with L226 in the HA protein displayed enhanced affinity for a2,6-sialoglycan and Sw/TZ exhibited affinity only for a2,6- sialoglycan. Interestingly, the viruses isolated from different hosts differed in their affinity for a2,3-sialoglycan. A pigeon-origin isolate, Pg/ XZ, had significantly decreased affinity for the a2,3-linked sialic acid, which may be associated with the predominant distribution of a2,6-linked sialic acid receptor in the pigeon respiratory tract [22]. The swine-origin isolate Sw/YZ displayed significantly decreased binding affinity for the avian-like receptor, and another swine-origin isolate, Sw/ TZ, had completely lost its affinity for the avian-like receptor. The receptor-binding preference may be altered after introduction into mammals [24], and these differences may reflect the viral adaptation to swine at different levels. The interaction between the HA protein of the influenza virus and its cellular receptor plays a critical role in regulating virus infection, replication and transmission [14, 36]. The MDCK cell line is commonly used for influenza A virus studies because it possesses abundant complex receptors (avian- and human-like receptors) and also supports efficient influenza virus replication. As expected, all
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H9N2 isolates replicated well in MDCK cells, although there were significant differences in titre among the isolates. The density of sialic acid receptors on cells is associated with the level of virus replication [25, 40]. However, in our study, the Sw/TZ virus, with the only human-like receptor specificity, was able to replicate well in DF-1 cells on which a2,3-linked sialic acid is predominantly expressed compared with a2,6-linked sialic acid [16]. Our experimental results together with previous findings [38] demonstrate that only a small amount of sialic acid is sufficient to support efficient replication of H9N2 viruses. A previous study demonstrated diverse replication phenotypes of H9N2 viruses in A549 cells [17]. Our growth kinetics assay also confirmed this, as evidenced by only three isolates, Ck/F98, Ck/TX and Sw/TZ, replicating in A549 cells, in which the complex receptors are well distributed. This phenomenon may be due to the HA-NA balance [41] or other factors, such as PB2 and NP [3, 11]. Previous studies have allowed the identification of a few critical molecular markers related to viral replication and pathogenicity. The molecular marker PB2 E627K is associated with enhanced viral replication ability in both mammalian cells and mice for H9N2 virus [20, 46]. The combination of PB2 D253N and Q591K [26] as well as the
Comparison of H9N2 AIVs
combination of HA A316S and a 3-amino-acid deletion in the NA stalk are also found to be correlated with high replication efficiency in cells and mice [33]. In our study, although all H9N2 viruses had PB2 627E combined with HA 316S (except for Ck/F98) and a short stalk in NA, they exhibited diverse replication phenotypes in A549 cells and mice, indicating that replication of these viruses is controlled by other unknown critical amino acids or gene constellations. A number of cell lines are widely used as in vitro models to mimic in vivo conditions. A recent study indicated that swine respiratory epithelial cells (SRECs) are an appropriate model system for examining the infectivity of swine-origin influenza viruses [5, 15]. In our study, we observed a similar replication pattern in both A549 cells and mice, because only the viruses that were propagated in A549 cells replicated in mouse lungs. Although virus replication in A549 cells does not fully reflect the level of infection in mice, A549 cells can be used initially to determine the potential pathogenicity of newly emerging AIVs to mammals prior to performing animal experiments. Although aquatic birds serve as a reservoir for influenza viruses, avian influenza viruses from aquatic birds must adapt to intermediate hosts prior to establishing stable lineages in land-based poultry [13, 31, 49]. Swine are regarded as good intermediate hosts, since they bear both a2,3- and a2,6-linked sialic acid receptors. In addition to swine, quail can also serve as an intermediate host for influenza viruses, due to the presence of both a2,3- and a2,6-linked sialic acid receptors in their respiratory tracts [42] as well as their susceptibility to influenza virus infection [4]. In addition to swine and quail, both a2,3- and a2,6-linked sialic acid receptors are present in the respiratory tract of chickens [9, 39]. In the present study, all H9N2 viruses derived from different hosts replicated and were transmitted efficiently in chickens without causing disease. These findings provide strong evidence that chickens provide a favourable environment for the circulation of H9N2 AIVs, which may lead to the increased possibility of reassortment of H9N2 AIVs with other cocirculating avian influenza viruses. This hypothesis is strongly supported by the recent outbreaks of H7N9 [48] and H10N8 [7], the internal gene segments of which are derived from H9N2 AIVs. A previous report indicated that a duck-origin influenza virus must adapt to quail and chickens via serial passage in their lungs to acquire efficient replicative ability in these species [13, 31]. However, in our study, the duck-origin virus Dk/Y33 replicated and was transmitted efficiently in chickens without prior adaptation in land-based species. Presumably, the H9N2 virus in China is likely to evolve to acquire enhanced interspecies transmissibility due to long-term circulation within poultry. Interestingly, both Dk/Y33 and Ql/A39 are
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sensitive to high temperature, whereas the other viruses are thermostable, which favors long-term persistence of H9N2 in the environment. Notably, both HA 363K and PA 672L of H9N2 viruses have been shown recently to be important for their airborne transmissibility among chickens [52]. Seven out of eight H9N2 viruses used in this study bore both HA 363K and PA 672L. They were likely to have airborne transmissibility in chickens. The high thermostability and efficient transmissibility of H9N2 viruses may increase the probability of transmission to susceptible hosts. Because of the pandemic potential of H9N2 AIVs and their contribution to the emergence of new reassortant viruses with the possible ability to cause a pandemic, we strongly recommended that we should strengthen the epidemiological surveillance for H9N2 avian influenza viruses and also broaden the range of surveillance hosts from common domestic poultry to swine, pigeons, and possibly wild aquatic birds. In conclusion, all of the tested H9N2 AIVs isolated from different hosts had a binding affinity for a2,6-linked sialic acid, but they varied in their binding affinity for a2,3linked sialic acid. However, all of the viruses displayed efficient replication and transmission properties in chickens. Our results highlight the important role played by chickens in the ecology and inter/intra-species transmission of H9N2 influenza viruses. Acknowledgments This study was supported by the Major State Basic Research Development Program of China (973 Program) (2011CB505003), the Important National Science and Technology Specific Projects (2012ZX10004214001002), the Sanxin Engineering Project for Jiangsu Agriculture (SXGC[2014]311), the Special Fund for Agro-Scientific Research in the Public Interest (201003012), the National High-Tech Research and Development Program of China (2011AA10A209), the Yangzhou University Funding for Scientific Research, and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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