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J Virol. 2016 November 1; 90(21): 9806–9825.
Published online 2016 October 14. Prepublished online 2016 August 24. doi:  10.1128/JVI.01141-16
PMCID: PMC5068531

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice

S. Schultz-Cherry, Editor
St. Jude Children's Research Hospital


H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs.

IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans.


Since the first isolate of an avian influenza virus (AIV) H9N2 subtype was reported in the United States in 1966, three distinct lineages of H9N2 viruses that caused outbreaks in domestic poultry in Asia have been identified (1, 2). H9N2 is a predominant subtype of AIVs circulating in poultry farms in Asia and the Middle East and has caused substantial economic losses over the past decade (3,9). Although a large amount of H9N2 vaccines, including inactivated and vectored vaccines, have been used in areas of endemicity, outbreaks caused by H9N2 AIVs are still not efficiently controlled. Importantly, H9N2 AIV has been reported to infect mammals, including humans, pigs, and dogs (10,13), indicating that it has an extended host range. It should be noted that several human infections with an H9N2 AIV have been recorded (14, 15). Numerous human infections have also been confirmed by serological survey, i.e., approximately 2.3 to 37.2% poultry workers in investigated farms are seropositive to the H9N2 virus (16,18). In addition, H9N2 AIVs have been shown to be donors of internal genes to generate zoonotic influenza viruses. For example, an H9N2 virus (A/quail/HK/G1/97) has contributed 6 internal genes to the highly pathogenic H5N1 influenza virus that claimed 18 victims in Hong Kong in 1997 (11). In 2013, two novel influenza viruses, H7N9 and H10N8, emerged sequentially after recruiting internal genes from H9N2 AIVs and caused human infections (19, 20). The H7N9 virus resulted in a >25% fatality rate in infected patients in China.

Receptor binding is the first step for the adaptation of AIVs to humans (21). Several studies showed that most of the recently isolated H9N2 AIVs in China have a mammalian type amino acid leucine (L) at position 226 in hemagglutinin (HA) in the receptor binding site that is responsible to bind to human type α2,6 sialic acid receptors (22, 23). To date, two amino acid residues, Q226L and I155T in HA protein, have been linked to receptor specificity changes in H9N2 AIVs (22,26). Further studies demonstrate that the H9N2 AIVs with these 2 amino acids can transmit among ferrets via aerosol droplets, suggesting that the H9N2 virus is a potential threat to mammalian hosts (23). Two other substitutions (N313D and N496S) in the HA of an H9N2 AIV have been shown to enhance binding to both α2,3-linked sialic acid (SAα2,3) and SAα2,6 receptors (27). Some amino acid mutations such as A316S in HA and M147L, D253N, A588V, Q591K, E627K, and D701N in PB2 have been identified to play a critical role in enhancing replication of AIVs in mammals (23, 28,31).

In this study, we isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets (LBMs) in China from 2009 to 2012. Thirty-seven representative isolates were selected based on phylogenetic analysis and geographic distribution for further characterization at the molecular level and in mice. Our results showed that some H9N2 viruses are able to replicate in mice efficiently, and further analysis demonstrated that the amino acid valine at position 190 in the HA (HA-190V) is responsible for efficient replication of H9N2 AIVs in mice.


Ethics statement.

All animal experiments were carried out in strict accordance with 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 protocol (Shvri-mo-0035) used in the study was approved by the Animal Care Committee of the Shanghai Veterinary Research Institute.

Sample collection and virus isolation.

A total of 12,032 oropharyngeal and cloacal swabs were collected from chickens and ducks in LBMs from different geographical areas in China, including Anhui, Fujian, Guangdong, Hunan, Jiangsu, Jilin, Shandong, Hebei, Shanxi, Hubei, and Zhejiang provinces and Shanghai municipality from 2009 to 2012 (Fig. 1). The collected samples were screened by using 9-to 11-day-old specific-pathogen-free (SPF) embryonated chicken eggs. The harvested allantoic fluids were tested by both hemagglutination assays and hemagglutination inhibition (HI) assays using a specific serum for each subtype of influenza A virus as described previously (32). The isolated viruses were stored at −80°C for future studies, and the isolated H9 viruses were further characterized in this study.

Geographical locations of H9N2 avian influenza viruses isolated in China.

Phylogenetic and molecular analyses.

Viral RNA was extracted from the harvested allantoic fluids that contained H9 subtype viruses, and each gene segment was amplified and sequenced as described previously (33). The nucleotide and deduced amino acid sequences were aligned and analyzed using the Megalign module of the Lasergene software (Dnastar, Madison, WI). Phylogenetic analyses were conducted using the Megalign program with the Clustal alignment algorithm and the neighbor-joining method (MEGA version 5.2). Estimates of the phylogenic relationships were calculated by performing 1,000 neighbor-joining bootstrap replicates.

Genotype definition.

Virus genotype was defined based on gene phylogenic analysis. The H9N2 viruses harboring G1-like genes were designated the genotype A series, while viruses harboring Ck/BJ/94-like genes were designated the genotype B series and further delineated as genotypes B1 to B65 as described previously (33,36). The analyzed sequences were derived from viruses isolated in this study or were downloaded from GenBank.

Antigenic analysis.

Based on the phylogenetic relationships of H9N2 influenza viruses, 19 representative viruses isolated during 2009 to 2012 and 3 early isolates (A/chicken/Shanghai/1/2006 [Ck/SH/1/06], A/chicken/Shanghai/6/2003 [Ck/SH/6/03], A/chicken/Shanghai/F/1998 [Ck/SH/F/98]) were selected for the antigenic analysis. Briefly, specific antisera against each isolate were produced in SPF chickens that were immunized with each inactivated virus with adjuvants. The HI assay was conducted to test cross-activity between each virus and serum as described previously (15). Numerical analysis of HI titers was performed using Primer version 7.0.7 (Primer-E, Plymouth, United Kingdom). The data were square-root transformed, and Bray-Curtis similarities were calculated. Two- and three-dimensional diagrams were also created using metric multidimensional scaling.

Plasmid construction and generation of single-mutated H9N2 AIVs.

The cDNAs of 8 gene segments of the H9N2 virus A/chicken/Jiangsu/A2093/2011 (A2093) or A/chicken/Zhejiang/B2013/2012 (B2013) were amplified by reverse transcription (RT)-PCR using the universal primers as described previously (37) and were cloned into the pBD plasmid (38). A single mutation (A190V, V190A, or V190T) in the HA gene of the A2093 or B2013 virus was generated using a site-directed mutagenesis kit (TransGene, Inc., Strasbourg, France). Wild-type A2093 and B2013 viruses as well as their single mutants were rescued as described previously (39) and were confirmed by sequencing.

Receptor binding assay.

The receptor preference of each virus was examined using a solid-phase binding assay as described previously (40). Streptavidin-coated clear strip plates (Thermo Fisher Scientific, Waltham, MA) were incubated with biotinylated glycans containing either α2,3-linked sialic acid (Neu5Aca2-3Galb1-4GlcNAcb-SpNH-PAA) or α2,6-linked sialic acid (Neu5Aca2-6Galb1-4GlcNAcb-SpNH-PAA) in washing buffer (phosphate-buffered saline [PBS] containing 1% bovine serum albumin [BSA] and 0.05% Tween 20) at 4°C overnight. Following 4 successive washes with the ice-cold washing buffer, the plates were inoculated with 32 HA units of influenza virus at 4°C overnight. The plates were washed 4 times with the washing buffer and then incubated with the chicken polyclonal antiserum against each H9N2 virus at 4°C for 2 h. After washing 4 times, the plates were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-chicken IgG antiserum (Sigma-Aldrich, St. Louis, MO) at 4°C for 2 h. Plates were washed 4 times and incubated with O-phenylenediamine (Sigma-Aldrich, St. Louis, MO), and then the reaction was stopped with 0.2 M H2SO4. The optical density in each well was determined at 490 nm using the Epoch Microplate Spectrophotometer (BioTek, Beijing, China).

Virus binding to mouse or human lung tissues.

The binding of the H9N2 viruses to normal mouse or human lung tissues was examined as described previously (40). In brief, paraffin-embedded normal mouse or human lung tissue sections (kindly provided by H. X. Chen, Hangzhou Hospital of Traditional Chinese Medicine, China) were deparaffinized and rehydrated. Tissue sections were then blocked by using 4% BSA in PBS and incubated with virus suspensions (64 HA units in PBS) at 4°C overnight. After being washed four times using the ice-cold PBS, the sections were incubated with the chicken polyclonal antibody against the H9N2 virus for 3 h at 4°C. The secondary antibody anti-chicken IgG conjugated with fluorescein isothiocyanate (FITC; 1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) was added. Sections were also counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Biyuntian, Shanghai, China) and examined by using confocal laser scanning microscopy (Nikon, Tokyo, Japan).

Real-time RT-PCR.

Confluent A549 cells (2.7 × 105 cells) were incubated with 64 HA units of each H9N2 virus diluted with PBS containing 4% BSA for 1 h at 4°C. After being washed four times in ice-cold PBS, cells were collected for RNA extraction using the RNeasy minikit (Qiagen, USA), and cDNA was generated using a uni-12 primer and reverse transcriptase AMV (TaKaRa, Dalian, China) according to the manufacturers' protocols. Real-time RT-PCR was performed in a 20-μl reaction mixture containing 10 μl premix Ex Taq (TaKaRa), 0.4 μl H9-AIV-Fw (CCATTGRACATRGCCCAG; 10 μM), 0.4 μl H9-AIV-Rw (CYATTTATTCGACTGTCGCCTC; 10 μM), 0.8 μl H9-AIV-probe (FAM-RGAAGGCAGGRAACCCCATTGCAA-TAQMAN-MGB; 10 μM), 2 μl RNA template, and 6.4 μl diethylpyrocarbonate (DEPC)-treated water. The assay was performed on a 7500 Real-time PCR instrument (Applied Biosystems) under the following conditions: 30 s at 95°C and 40 cycles of 5 s at 95°C and 34 s at 60°C. Tenfold serial dilutions of the plasmid pMD19T-H9HA, which contains an H9N2 HA gene, were used as the templates for real-time RT-PCR to generate the standard curve for determination of virus copies. Each sample assay was repeated 3 times.

Flow cytometry analysis.

Confluent A549 cells (2 × 106 cells) were incubated with 1,024 HA units of each H9N2 virus diluted with PBS containing 4% BSA for 1 h at 4°C and then incubated with the chicken polyclonal antibody against each H9N2 virus for 1 h at 4°C and finally with a secondary antibody anti-chicken IgG conjugated with FITC. Flow cytometry was performed on the FACSCanto flow cytometers (BD), and the mean fluorescence intensity (MFI), which represents the quantity of virus proteins, was analyzed using the FlowJo (Tree Star, Inc.). For negative controls, A549 cells were incubated only with the secondary antibody without virus binding, or A549 cells were incubated only with both the chicken polyclonal antibody against H9N2 virus and the secondary antibody without virus binding.

Mouse experiments.

Thirty-seven representative H9N2 isolates were selected for the mouse study based on the HA gene phylogenetic analysis. Groups of 9 6-week-old BALB/c mice were slightly anesthetized with CO2 and inoculated intranasally with 106 50% egg infective doses (EID50) of each H9N2 virus in a volume of 50 μl. Clinical signs and weight loss were monitored daily. Three mice in each group were euthanized at 4 days postinfection (dpi), and the lungs were collected for virus titration. The remaining mice were kept for 14 days, and serum samples were collected at the end of the experiment.

Statistical analysis.

Virus titers, RNA copy numbers, and protein quantity data between groups were analyzed using analysis of variance (ANOVA) in the GraphPad Prism software (GraphPad Software Inc.); a P value of 0.05 or less was considered significant. The response variables shown to have a significant effect by treatment group were subjected to comparisons for all pairs by using the Tukey-Kramer test. Pairwise mean comparisons between inoculated and control groups were made using the Student t test.

Accession number(s).

The nucleotide sequences obtained in this study are available in GenBank under accession numbers KP865753 to KP866100.


Virus isolation and genotype characterization.

A total of 12,032 swab samples were collected from healthy chickens (6,903 samples) and ducks (5,129 samples) in LBMs located in 12 different provinces in China from 2009 to 2012 (Fig. 1 and Table 1); different subtypes of influenza A viruses, including H3, H4, H6, H7, H9, H10, and H11, were detected. A total of 989 H9N2 AIVs were recovered and further characterized in this study. The majority of H9N2 viruses (87%, 865/989) were isolated from chicken samples, and 13% (124/989) of viruses were from duck samples. The isolation rates from chickens in 2009, 2010, 2011, and 2012 were 14.20%, 11.90%, 15.18%, and 8.20%, respectively, while from ducks they were 2.85%, 6.32%, 1.63%, and 1.35% in each respective year (Table 1). Of 989 H9N2 AIV isolates, 37 representative isolates were selected based on geographical and host differences to perform full-genome sequencing and further analysis, including 11 viruses isolated in 2009, 12 viruses isolated in 2010, 7 viruses isolated in 2011, and 7 viruses isolated in 2012.

Prevalence of H9N2 viruses from chickens and ducks in China from 2009 to 2012

Phylogenetic analysis showed that the HA genes of selected 37 H9N2 isolates belonged to the Ck/BJ/1/94-like lineage that includes two subgroups, I and II (Fig. 2A). All HA genes of 37 selected H9N2 viruses isolated during 2009 to 2012 clustered into subgroup II and were distinct from those of early H9N2 isolates in China, which are grouped into subgroup I. The NA genes of all selected H9N2 AIVs were clustered into the Ck/BJ/1/94-like lineage (Fig. 2B).

Phylogenetic analysis for HA, NA, PB2, PB1, PA, NP, M, and NS genes and the antigenicity of H9N2 avian influenza virus isolates. The phylogenetic trees for each gene were generated by the neighbor-joining method in MEGA version 3.1. The trees were rooted ...

Based on the phylogenetic analysis of all 8 gene segments of 37 H9N2 AIVs (Fig. 2A to toH),H), 5 novel genotypes of H9N2 viruses (B65 to B69) were identified in 2009 isolates, and another 2 new genotypes (B70 and B71) were found in 2010 isolates (Fig. 3). No new genotype was found during the 2011-2012 surveillance. Interestingly, the genotype B69 virus appeared in 2009, and subsequently it has become a dominant virus in poultry, including chickens and ducks, in China since 2010.

Genotypes of H9N2 avian influenza viruses isolated from chickens and ducks in China. In the ovals, horizontal bars from top to bottom stand for PB2, PB1, PA, HA, NP, NA, M, and NS. Abbreviations: Ck/BJ/94, A/Chicken/Beijing/1/1994 (H9N2); Ck/HK/G9/97, ...

Molecular and antigenic characterization of isolated H9N2 viruses.

Five H9N2 AIVs isolated by our laboratory prior to 2006 possessed 226Q in the HA, which is an avian type amino acid at the receptor binding site, while all 37 viruses isolated during 2009 to 2012 contained 226L in the HA, which is a human type amino acid at the receptor binding site (Table 2). Most of the H9N2 viruses had a PSRSSR motif at the connecting peptide between their HA1 and HA2 subunits, 3 H9N2 viruses isolated in 2009 had a PSKSSR motif, and the isolate A/chicken/Shanghai/340/2009 had PSISSR at the HA cleavage site. Two H9N2 viruses isolated in 2009 and 2010 had a PARSSR motif, which is identical to those of early H9N2 isolates before 2006 (Table 2). The isolate B2013 had a PSRSNR motif at the HA cleavage site. All facts indicate that all H9N2 isolates were low-pathogenic AIVs. Most of H9N2 isolates had a three-amino-acid deletion at positions 62 to 64 in the NA stalk region, while 3 viruses belonging to genotypes 70 and 71 lacked this deletion and had a full-length stalk similar to those of early H9N2 isolates (Table 2). Regarding the amino acids associated with amantadine resistance on the M2 protein (1), one early isolate, Ck/SH/1/06, had a V27A mutation, and two recent isolates, A/chicken/Guangdong/1494/2009 and A/chicken/Fujian/A1166/2010, had a V27I substitution. More than one-half of the viruses isolated during 2009 to 2012 had a 31N, and the percentage of S31N substitution has increased with time (Table 2), suggesting a wide spread of amantadine-resistant H9N2 AIVs. All of the H9N2 viruses contained avian type related residues 627E and 701D in their PB2 proteins (41, 42).

Amino acid residues in HA, NA, M2, and PB2 of H9N2 AIVsa

Crossing HI tests were conducted to investigate the antigenic properties of the H9N2 isolates. A vaccine strain, Ck/SH/F/98 (43), and 2 early isolates (Ck/SH/1/06, Ck/SH/6/03) were also used for this assay. Based on the crossing HI titers of each pair of viruses (Fig. 2I), the H9N2 viruses isolated during 2009 to 2012 formed a novel antigenic subgroup that was separated from the subgroup of early virus isolates, except for the A/chicken/Jilin/3/2009 (CK/JL/3/09) virus, whose antigenicity is close to that of the early isolate Ck/SH/1/06.

Replication of H9N2 viruses in mice.

To determine replication capability of selected 37 H9N2 isolates in mammals, groups of mice were inoculated intranasally with 106 EID50 of each virus. No obvious clinical signs, including weight loss, ruffled fur, and decreased activity, were observed in any group of infected mice compared to the controls. The replication capabilities of 37 tested viruses in mice were variable. All infected mice seroconverted at the end of the experiment with an HI titer ranging from 20 to 640. The tested B64 (5 isolates), B66 (2 isolates), and B70 (1 isolate) genotypes of viruses were not detectable in mouse lungs (Fig. 4) despite the occurrence of seroconversion. Two viruses in genotype B68 propagated in mouse lungs, but the virus was detected in only 1 of 3 infected mice with a lower titer. In contrast, some of the tested viruses in genotypes B69 (12/25) and B71 (1/2) replicated in mouse lungs. Interestingly, only 3 viruses of 37 isolates, including A/chicken/Shanghai/3392/2010, A2093, and A/duck/Anhui/C313/2012, in genotype B69 replicated efficiently in mouse lungs, and they were detected in all 3 infected animals with average titers of 104.75, 103.92, and 105.00 EID50/ml, respectively. The results indicate that variation of replication capability existed among H9N2 AIVs in mice (Fig. 4), and most likely several H9N2 viruses might already start to adapt to mammalian hosts.

Virus titers in lungs of mice infected with different H9N2 AIVs. Mice were intranasally infected with 106 EID50 of each H9N2 virus. Three mice of each group were euthanized at 4 days postinoculation, and lung was collected for virus titration. Virus titers ...

HA-190V is responsible for enhanced replication of H9N2 AIVs in mice.

To determine why these 37 H9N2 isolates showed different replication capability in mice, we focused on analyzing the differences in PB2 and HA of theses isolates because these two proteins of AIVs have been demonstrated to play crucial roles in cross-species infection of mammals (23, 27, 29, 30, 42). For the PB2 protein, all these isolates had avian type residues 627E and 701D, which are critical for mammalian adaptation (Table 2). These 37 viruses had the same amino acids at the HA receptor binding site (183N, 226L, and 228G). Noticeably, these viruses had a different amino acid at position 190 in the HA (H3 numbering): 27 isolates with 190A (alanine), 6 viruses with 190T (threonine), and 4 viruses with 190V (valine). More than one-half of viruses with HA-190A (18/27) could not be detected in the inoculated mice at 4 dpi, and the remaining viruses were only detected in 1 (8/27) or 2 (1/27) of 3 infected animals (Fig. 4). The viruses with HA-190T were detected in 1 or none of 3 infected mice. Interestingly, all 3 viruses replicated efficiently in mouse lungs that had HA-190V, whereas virus CK/JL/3/09 with HA-190V replicated poorly in mice.

To determine whether HA-190V plays a critical role in enhancing virus replication in mice, both A2093, which had an HA-190V and replicated efficiently in mouse lungs, and B2013, which possessed an HA-190T and could not replicate in mouse lungs, were selected to generate single-mutated viruses (A2093-HA-V190A, A2093-HA-V190T, and B2013-HA-T190V) by using reverse genetics. Both rescued A2093 (r-A2013) and rescued B2013 (r-B2013) kept replication properties in mice similar to those of their parental viruses (Fig. 5). The r-B2013-HA-T190V virus with T190V substitution in HA replicated efficiently in mice compared to both the wild-type B2013 (wtB2013) and r-B2013. Substitution of V with A or T at position 190 in the HA (A2093-HA-V190A, A2093-HA-V190T) resulted in no replication of the A2093 virus in mice. These data indicate that HA-190V is responsible for enhanced replication of H9N2 AIVs in mice.

Replication of rescued wild-type A2093 or B2013 and their single-mutated viruses in mouse lungs. Wild-type A2093 or B2013 and their single-mutated (HA-190A, T, or V) viruses were rescued. Mice were intranasally infected with 106 EID50 of each virus, and ...

HA-190V does not affect receptor binding specificity but increases virus attachment to mouse and human lung tissues.

To understand why and how the residue in position 190 in HA influences virus replication in mice, we first investigated whether this specific amino acid affects the specificity of virus receptor binding, which is the first step of influenza virus infection (44). A solid-phase binding assay using biotinylated glycans containing either α2,3-linked or α2,6-linked sialic acids was performed to determine virus receptor binding specificity of both the rescued wild-type viruses and their single-mutated viruses (r-A2093, r-A2093-V190T, and r-A2093-V190A; r-B2013 and r-B2013-T190V). The results showed that all these 5 viruses preferentially bound to the α2,6-linked sialic acid receptors (Fig. 6), indicating that the residue at position 190 in the HA does not influence virus receptor binding specificity.

Receptor binding specificity of different H9N2 viruses. The receptor-binding properties of wild-type H9N2 viruses and their single mutants were determined by a solid-phase binding assay. A recombinant virus with HA and NA genes from the 2009 pandemic ...

To determine whether the residue at position 190 in the HA influences virus binding affinity, we performed the virus binding assay using both mouse and human lung tissues. Both mouse and human lung tissues contain the SAα2,6 glycans mainly (45, 46). The results revealed that r-A2093 showed a significantly higher binding to both human and mouse lung tissues than r-B2013. The single substitution T190V significantly enhanced binding of r-B2013 to both human and mouse lung tissues, i.e., the r-B2013-T190V virus displayed much higher binding ability to lung tissues than the r-B2013 virus. In contrast, the single substitution V190T (r-A2093-V190T) or V190A (r-A2093-V190A) in the HA of r-A2093 resulted in poor binding or demolished the binding to both mouse and human lung tissues (Fig. 7). These results indicated that HA-190V affects binding affinity, not receptor binding specificity, resulting in enhanced virus replication in mouse lungs.

Binding of parental and single-mutated H9N2 viruses to mouse and human lung tissues. Lung sections were incubated with each H9N2 virus and subsequently incubated with an anti-H9N2 polyclonal antibody and an FITC-labeled secondary antibody and DAPI dye ...

HA-190V increases virus binding to A549 cells.

In order to precisely compare binding abilities among the viruses with HA 190V/A/T, we first detected the RNA copy numbers after the H9N2 viruses were incubated with the A549 cells for 1 h. The RNA copies reached 1.4 × 106 copies in r-A2093 bound to A549 cells (Fig. 8A). When HA-190V was mutated to 190A (r-A2093-V190A) or 190T (r-A2093-V190T), the quantities of RNA copies were decreased by 90.7% or 78.3%, respectively. Similarly, when HA-190T (r-B2013) was mutated to 190V (r-B2013-T190V) in the genetic background of B2013, the quantity of RNA copies was significantly increased (>6-fold) compared to the parental r-B2013.

Viral RNA copies and viral protein amounts in A549 cells bound with different H9N2 viruses. A549 cells were incubated with each H9N2 virus for 1 h and then washed 3 times. The treated cells were used to extract RNA to quantify viral RNA copies using a ...

In order to confirm that HA-190V enhances virus binding to the A549 cells, we performed flow cytometry analysis to determine virus binding amounts at the protein level and used the MFI value to represent the quantity of virus protein binding to the A549 cells. As shown in Fig. 8B, when HA-190V (r-A2093) was mutated to 190A (r-A2093-V190A) or 190T (r-A2093-V190T), the MFI values were reduced by 21.8% or 20.2% compared to the parental r-A2093. In contrast, the MFI value of r-B2013-T190V significantly increased by 52.5% compared to the parental r-B2013 with HA-190T (Fig. 8C).

Prevalence of HA-190V in H9N2 viruses.

To determine the prevalence of HA-190V mutations occurring naturally in H9N2 viruses, available H9N2 HA sequences were analyzed. The proportion of H9N2 viruses harboring an HA 190V was approximately 11.90% before 1999, but it increased to 22.00% in the following 3 years (2000 to 2002) after the first reported human H9N2 infection in 1999. During 2003 to 2007, the average rate of H9N2 viruses having an HA-190V was approximately 12.5%. Starting at 2008, the proportion of H9N2 viruses with an HA-190V increased to more than 21.00% in the following 5 years (Table 3). The results indicate that the H9N2 virus containing HA-190V has been established over the past decade. Importantly, to date 66.70% (6/9) of human H9N2 isolates whose sequences are available in GenBank harbored HA-190V (Table 4).

Percentages of H9N2 viruses with each amino acid at position 190 in the HA in different yearsa
Natural mutations at position 190 in the HA of H9N2 viruses that have infected humans


H9N2 virus is one of the major subtypes of influenza viruses circulating in domestic poultry worldwide, and it causes significant economic losses to poultry industries and also threatens public health. Our surveillance in LBMs in China during 2009 to 2012 shows that the H9N2 virus was prevalent in chickens; this result is consistent with the finding in a former report that was conducted in farm chickens in China (47). All these results indicate that the H9N2 virus is becoming a predominant virus circulating in domestic poultry in China. Noticeably, the recently identified H9N2 viruses are distinct from the vaccine strains used, indicating that the virus changes very fast, especially the HA gene due to the immunity and selection pressure (47). Rapid antigenic drift and aerosol transmission among birds have been major issues in the attempts to control the H9N2 avian influenza and could accelerate the spread and evolution of H9N2 AIVs (48).

In previous reports, genotype B series of H9N2 viruses have been identified as having become prevalent primarily in domestic poultry in China since the mid-1990s (11, 35, 49). Seven novel genotypes (B65 to B71) of H9N2 viruses appeared and were identified during 2009 to 2013. Six of these genotypes existed for 1 year and then disappeared; only genotype B69 survived, and viruses of this genotype have become dominant since 2011 (47). Based on our surveillance data, these 7 genotypes of H9N2 viruses were also found in LBMs, but genotype B69 has been predominant in chickens and ducks starting in 2010. This genotype H9N2 virus has displayed more efficient replication in chickens than viruses of other genotypes due to improved viral fitness through antigenic changes (47). All characterized 37 H9N2 isolates belonging to 6 different B genotypes have an HA 226L that is responsible to bind to human type α2,6 sialic acid receptors; this fact is consistent with findings of previous studies (25, 50). Interestingly, only three H9N2 isolates in genotype B69 identified in this study replicated efficiently in mice, indicating that these viruses adapt to infect mammals, in contrast to other viruses tested in parallel. In addition, the H9N2 viruses in this genotype have been demonstrated to provide internal genes for zoonotic emerging H7N9 and H10N8 viruses that have infected humans and caused fatality in infected patients (19, 20), indicating that this genotype of virus has a high genetic compatibility with other influenza A viruses.

Infections of mammals such as humans and pigs with an H9N2 virus have been well documented (14, 51,53). Specific binding of human type receptors is the first step to enable an H9N2 AIV to cause cross-species infection of mammals. The H9N2 AIVs that harbor Q226L and I155T in HA have been reported to preferentially recognize the human type SAα2,6 receptors (22, 23). In this study, we demonstrate that HA-190V is responsible for efficient replication of H9N2 viruses in mice. Furthermore, the amino acids (A/T/V) at position 190 in the HA did not affect H9N2 virus receptor binding specificity, and these viruses still specifically bound to human type SAα2,6 receptors. This result is consistent with the previous report that H9N2 viruses harboring HA-190A bind to SAα2,6 receptors (23).

Although the T190V, V190T, and V190A substitutions in HA did not change the binding specificity of tested H9N2 AIVs to SAα2,6 receptors, those mutations did significantly affect HA affinity and attachment to cells and lung tissues from mouse and humans. Many different glycan structures presented on SAα2,6 receptors were not available in the substrates used in our receptor binding assays. Thus, the affinity of wild-type and single-mutated influenza viruses to SAα2,6 receptors was directly tested on human A549 cells and on lung tissues from mouse and human (44, 46). When V was present at position 190 in the HA, both r-A2093 and r-B2013-T190V showed firmer attachment to human A549 cells than did each respective virus with HA-190T or HA-190A, resulting in detection of significantly greater numbers of viral RNA and antigens. Both viruses also displayed efficient binding abilities in mouse and human lung tissues. All these results indicate that HA-190V is critical for enhanced virus binding affinity. Other mutations in the HA of H9N2 AIVs, such as S127N and T198N, have been shown to affect viral virulence in mammals (54). In this study, HA-190V of the genotype B69 H9N2 viruses has been shown to be critical for virus replication in mice. However, an H9N2 virus belonging to the genotype B66 has 190V in the HA and cannot replicate in mouse lungs. These facts suggest that the HA-190V facilitates virus infection of H9N2 viruses in mice but synergetic effects of other critical amino acids are most likely needed. A former study has shown that the D190E substitution in combination with changes at position 193 resulted in a 10-fold increase in the 50% lethal dose (LD50) of virulent PR8 H1N1 virus (55). Similarly, the H5N1 viruses with a double mutation at HA residues 129 and 151 has increased infectivity and binding affinity to the human lower respiratory tract (56). So far, we do not know which amino acid(s) commits to synergetic effects with the HA-190V; this will be investigated in future studies.

Taken together, our results indicate that the amino acid V at position 190 in HA does not affect virus receptor binding specificity but significantly enhances HA affinity to human cells and virus attachment to mouse and human lung tissues, thereby resulting in increased virus replication in mice. Based on the findings in our study and the fact that the H9N2 virus has infected humans (14, 15) and started to adapt to mammals (13, 23, 27, 28), more attention should be paid to the H9N2 viruses possessing HA-190V, which might have abilities to cross-species infect mammals, including humans.


This study was supported by the National Natural Science Foundation of China (31472206 and 31302115) and the National Key Research and Development Plan (2016 YFD0500204).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.


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