Expression of influenza B virus hemagglutinin and its immunogenicity determination / 生物工程学报

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

Characterization of a monoclonal antibody against the hemagglutinin stem of H7N9 subtype avian influenza virus / 生物工程学报

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (431W-433Y-437L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.

High expression and immunogenicity analysis of HA globular head domain of H5 subtype avian influenza virus produced in Pichia pastoris / 生物工程学报

To evaluate the immunogenicity of HA globular head domain of H5 subtype influenza virus (H5HA), the gene of H5HA was optimized and the recombinant pPICZaA-H5HA expressing vector was constructed and transfected into Pichia pastoris. The expression of the recombinant H5HA was confirmed by SDS-PAGE and Western blotting and the results demonstrated that the recombinant H5HA (37 kDa) was highly expressed in Pichia pastoris with concentration of 0.2 mg/mL in medium. The recombinant H5HA was concentrated and purified using Ni-NTA affinity chromatography. The immunogenicity of H5HA was evaluated by immunizing eight groups of chicken through intranasal or intramuscular injection with different doses of purified H5HA combined with different adjuvants, respectively. The results showed that the recombinant H5HA could induce high level IgG (HI titer was 1:64 and neutralizing antibody titer was 1:218) and the optimal dosage of the recombinant H5HA was 50 μg combined with oil. In addition, intramuscular injection was better than nasal immunization. This study provided a theoretical support for subunit vaccine development.

Evolution of equine influenza viruses (H3N8) during a Brazilian outbreak, 2015

Abstract Equine influenza is one of the major respiratory infectious diseases in horses. An equine influenza virus outbreak was identified in vaccinated and unvaccinated horses in a veterinary school hospital in São Paulo, SP, Brazil, in September 2015. The twelve equine influenza viruses isolated belonged to Florida Clade 1. The hemagglutinin and neuraminidase amino acid sequences were compared with the recent isolates from North and South America and the World Organisation for Animal Health recommended Florida Clade 1 vaccine strain. The hemagglutinin amino acid sequences had nine substitutions, compared with the vaccine strain. Two of them were in antigenic site A (A138S and G142R), one in antigenic site E (R62K) and another not in antigenic site (K304E). The four substitutions changed the hydrophobicity of hemagglutinin. Three distinct genetic variants were identified during the outbreak. Eleven variants were found in four quasispecies, which suggests the equine influenza virus evolved during the outbreak. The use of an out of date vaccine strain or updated vaccines without the production of protective antibody titers might be the major contributing factors on virus dissemination during this outbreak.

Identification of Epitopes for Neutralizing Antibodies Against H10N8 Avian Influenza Virus Hemagglutinin / 中国医学科学院学报

Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection.

Preparation and Identification of High Immunogenic A/PR/8/34 Maternal Strain HA Protein for Influenza Virus Classical Reassortment / 病毒学报

Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China.

Virological characteristics of influenza A (H3N2) virus in mainland China during 2013-2014 / 病毒学报

To analyze the antigenic and genetic characteristics of the influenza A (H3N2) virus in mainland China during the surveillance year of 2013-2014, the antigenic characteristics of H3N2 virus were analyzed using reference ferret anti-sera. The nucleotide sequences of the viruses were determined by Sanger dideoxy sequencing, phylogenetic trees were constructed with the neighbor-joining method, and the genetic characteristics of the viruses were determined in comparison to current vaccine strains. The results showed that most of the H3N2 viruses were antigenically closely related to the A/Victoria/361/2011 vaccine strain cell-propagated prototype virus (99.6%). Using the A/Texas/50/2012 egg isolate as the reference antigen, 15.1% of the viruses were found to be closely antigenically related to it, while 11.9% of strains were closely antigenically related to the egg-propagated epidemic strain, A/Shanghai-Changning/1507/2012. Phylogenetic analysis of HA genes indicated that the A(H3N2) viruses in this surveillance year were in the same clade, but no drug resistant mutation was identified in the NA genes. During the 2013-2014 influenza surveillance year, no significant genetic change was detected in either the HA or NA genes of the A(H3N2) viruses, while significant mutations were found in egg isolates resulting from their adaptation during propagation in eggs. The antigenic and genetic changes should be investigated in a timely manner to enable the selection of an appropriate vaccine strain in China.

Isolation and phylogenetic analysis of hemagglutinin gene of H9N2 influenza viruses from chickens in South China from 2012 to 2013

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSRdownward arrowGLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSRdownward arrowGLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.

Expression of Chimeric Influenza Hemagglutinin Antigen (cH7/3) using the Baculovirus Expression System and Identification of its Biological Activities / 病毒学报

Hemagglutinin (HA) contains a head domain with a high degree of variability and a relatively conserved stem region. HA is the major viral antigen on the surface of the influenza virus. To define the biologic activities of chimeric HA bearing different head domains and stem regions or their potential use, a HA chimeric gene containing the head domain of the H7 subtype virus and stem region of the H3 subtype virus was modified and expressed using a baculovirus expression vector. Then, the secreted protein was purified and its biologic activities characterized. Approximately 1.4 mg/mL cH7/3 HA could be obtained, and its molecular weight was ≈ 70 kD. The trimer form of cH7/3 protein had hemagglutination activity and could be recognized by specific antibodies. The method described here can be used for further studies on the screening of HA stem-reactive antibodies or the development of vaccines with conserved epitopes.

Analysis of HA and NA Genes of Influenza A H1N1 Virus in Yunnan Province during 2009-2014 / 病毒学报

To analyze influenza pathogen spectrum in Yunnan province during 2009-2014 years, and analyze HA and NA genes of influenza A H1N1. Analysis was made on the monitoring date of influenza cases in Yunnan province in recent 6 years, 23 strains of influenza virus of HA and NA gene was sequenced and analyzed by MEGA 5 software to construct phylogenetic tree. 4 times of influenza AH1N1 epidemic peak were monitored from 2009-2014 years in Yunnan Province, as the nucleic acid detection results of influenza A H1N1 accounted for 28.8% of the total. The sequencing result showed that HA and NA gene were divided into 3 groups, one was detected with H275Y mutation strains. Influenza A H1N1 is one of the important subtypes in Yunnan province and their genes have divided into three branches during the period of 2009-2014 years, the vast majority of influenza a H1N1 are still sensitive to neuraminidase inhibitors.

Preliminary study of a universal vaccine based on the HA2 protein of the H5N1 influenza virus / 病毒学报

Fragments encoding amino acids 76-130 in the linear conserved region (LCR) of A/Hubei/1/2010 (H5N1) HA2 was fused to hepatitis B core antigen (HBc) to generate a LCR-HBe virus-like particle (VLP). Results showed that the fusion protein of LCR-HBc was highly expressed in this prokaryotic expression system. The purified LCR-HBc particle stimulated high levels of IgG production in mice with a titer of > 1:12 800, and provided 50% cross-protection against lethal challenge by H1N1 viruses.

Adaptive evolution of the hemagglutinin genes of the H6N1 avian influenza virus in Taiwan, China / 病毒学报

In Taiwan, the first human-infecting H6N1 avian influenza virus was isolated in 2013. To better understand the origin, evolutionary relationship and pathogenesis of the H6N1 virus, we studied the adaptive evolution and evolutionary dynamics of the hemagglutinin (HA) genes of the H6N1 virus in Taiwan. We felt that such studies woud contribute to the further study and control of the virus. Datasets were gained from the Flu and Global Initiative on Sharing All Influenza Data (GISAID) databases. Then, phylogenetic trees and evolutionary dynamics were reconstructed. The evolutionary rate and characterization of adaptive evolution were analyzed by bioinformatic methods. Results indicated that the HA genes of H6N1 in Taiwan were divided into at least five types, and that the new types that the infected human H6N1 belonged to could be local advantage type at present. Evolutionary dynamics revealed the viral population expanded first at the end of 1971, reduced sharply in 2008, and then increased slightly. Three sites were identified under positive selection, suggesting that various sites might increase the adaptive ability of the virus. Eighty-nine sites were under negative selection, revealing that these sites might play an important role in the replication and epidemiology of the virus. Interestingly, site 329 upstream from the cleavage site was also under negative selection, suggesting that this site might be associated with the virulence of H6N1. These data suggest that the HA genes of the Taiwanese H6N1 virus have been undergoing adaptive evolution, and that an outbreak may occur again. Hence, more attention should be paid to the identified sites, to enable timely monitoring and control of a future epidemic.

Construction and identification of helper-dependent adenoviral vector encoding hemagglutinin protein of swin-origin influenza virus A H1N1 / 生物医学工程学杂志

Abstract: In order to investigate immune protection against swine-origin influenza virus (S-OIV) A H1N1, the helper-dependent adenovirus vector (HDAd) system was exploited to construct recombinant HDAd encoding hemagglutinin (HA). The HA gene was synthesized and cloned to the HDAd backbone. Then, the HDAd/HA DNA molecules were transfected into 293Cre4 cells with calcium phosphate. The cells were infected by helper virus 16 hours after the transfection. The 293Cre4 cells were coinfected with HDAd/HA and the helper virus for large-scale preparation of HDAd/HA. The HDAd/HA was obtained and purified twice with CsCI density ultracentrifugation and observed morphologically under transmission electron microscope, and the expression of HA protein was analyzed with RTPCR. Recombinant HDAd/HA expressing HA protein was successfully constructed which could pave the way for in vivo investigation on immunogenicity and efficacy against S-OIV A H1N1 infection.

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.</p><p><b>METHODS</b>Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses.</p><p><b>RESULTS</b>The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.</p><p><b>CONCLUSION</b>The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.</p>

Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system / Expresión de la subunidad HA1 del virus de influenza equina utilizando un sistema de expresión en baculovirus

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20 μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests.

El virus de la influenza equina es una de las principales causas de enfermedad respiratoria en caballos de todo el mundo. La prevención de la enfermedad es a través de la vacunación con vacunas a virus inactivado. La mayoría de las vacunas se producen en huevos embrionados, de los cuales los viriones son cosechados del líquido alantoideo e inactivados químicamente. Aunque este sistema ha servido bien durante años, el uso de huevos como sustrato para la producción de vacuna presenta varias desventajas bien reconocidas (costo, provisión de huevos, manejo de los residuos, rinde por huevo). El objetivo del presente trabajo fue evaluar preliminarmente un sistema de expresión en baculovirus como método de producción de hemoaglutinina recombinante (rHA) para ser utilizada como vacuna para la prevención de la influenza equina. Para ello el ectodominio de la hemaglutinina (la subunidad HA1) del virus de la influenza equina se expresó en células de insecto infectadas con un baculovirus recombinante. La expresión fue demostrada por SDS-PAGE e inmunoblotting. El método empleado fue capaz de producir gran cantidad de rHA1. En este estudio se obtuvieron 20 μg/ml (200 μg de HA1 purificada de 2,5x107 células infectadas). La respuesta inmune fue evaluada mediante la inmunización de ratones BALB/c. Los resultados preliminares demostraron que la proteína recombinante expresada en baculovirus genera una fuerte respuesta inmune en ratones, por lo tanto podría ser utilizada como antígeno para la producción de una vacuna a subunidades y en pruebas diagnósticas.

Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut)

Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka's group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.

Origin and molecular characterization of the human-infecting H6N1 influenza virus in Taiwan

In June 2013, the first human H6N1 influenza virus infection was confirmed in Taiwan. However, the origin and molecular characterization of this virus, A/Taiwan/2/2013 (H6N1), have not been well studied thus far. In the present report, we performed phylogenetic and coalescent analyses of this virus and compared its molecular profile/characteristics with other closely related strains. Molecular characterization of H6N1 revealed that it is a typical avian influenza virus of low pathogenicity, which might not replicate and propagate well in the upper airway in mammals. Phylogenetic analysis revealed that the virus clusters with A/chicken/Taiwan/A2837/2013 (H6N1) in seven genes, except PB1. For the PB1 gene, A/Taiwan/2/2013 was clustered with a different H6N1 lineage from A/chicken/Taiwan/ A2837/2013. Although a previous study demonstrated that the PB2, PA, and M genes of A/Taiwan/2/2013 might be derived from the H5N2 viruses, coalescent analyses revealed that these H5N2 viruses were derived from more recent strains than that of the ancestor of A/Taiwan/2/2013. Therefore, we propose that A/Taiwan/2/2013 is a reassortant from different H6N1 lineages circulating in chickens in Taiwan. Furthermore, compared to avian isolates, a single P186L (H3 numbering) substitution in the hemagglutinin H6 of the human isolate might increase the mammalian receptor binding and, hence, this strain's pathogenicity in humans. Overall, human infection with this virus seems an accidental event and is unlikely to cause an influenza pandemic. However, its co-circulation and potential reassortment with other influenza subtypes are still worthy of attention.

Genetic variation of the hemagglutinin and neuraminidase of influenza B viruses isolated in Ningbo during 2010-2012 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the epidemiological characteristics of influenza B viruses and explore the genetic evolution characteristics of the hemagglutinin(HA) and neuraminidase(NA) genes of local isolated strains in Ningbo, Southeast China, during 2010 to 2012.</p><p><b>METHODS</b>Respiratory specimens from 3440 cases of patients with influenza-like illness(ILI) during 2010 to 2012 were collected in for virus isolation. And the 628 sera samples were collected in 2010 from the healthy community population to detect the HI antibody level against the local isolated virus.For phylogenetic analysis, the fragments of HA and NA genes were amplified and sequenced from strains isolated in different years. The association between evolution of HA and epidemiological characteristics were analyzed.</p><p><b>RESULTS</b>A total of 109 strains of influenza B virus were isolated, including 102 (93.6%) Victoria-lineage strains and 7 (6.4%) Yamagata-lineage strains. Positive rates of HI antibody against Victoria-lineage strains and Yamagata-lineage strains were 51.1% (321) and 47.8% (300), respectively (χ(2) = 1.405, P > 0.05) among the 628 sera samples. The phylogenetic analysis showed that all HA fragments of isolated strains clustered the same branch with Malaysia/2506/2004 while the NA genes formed different branches. Compared with Brisbane/60/2008 strain, there were 1 to 5 Amino acid mutations in HA domain, and more mutations were detected in NA domain, ranged from 6 to 16 sites. The genetic evolution of NA in Victoria-lineage strains were faster compared with HA.</p><p><b>CONCLUSION</b>The genetic evolution rates of NA genes were faster than that of HA genes in the local circulated Victoria-lineage viruses during 2010 to 2012;The comprehensive analysis of HA and NA fragments were more reliable and sensitive on surveillance of genetic evolution of influenza B viruses.</p>

Inhibition of proliferation of H5N1 subtype AIV in CEF by chemosynthetic siRNA / 病毒学报

In order to study the proliferation inhibition effect of H5N1 subtype avian influenza virus (AIV) with small interfere RNA (siRNA), a total of 4 siRNAs were designed in accordance with the NP and PA genes of H5N1 subtype AIV, the siRNAs were then transfected to chicken embryo fibroblast(CEF), CEF was infected with H5N1 subtype AIV after 6 hrs. Virus titer of cell supernatant was tested at 16-56hrs post infection, and pathological changes of the cells was observed; mRNA levels of NP, PA, HA and p13-actin gene were tested at 36hrs post infection. The results showed that these 4 siRNAs could inhibit the prolif-eration of H5N1 subtype AIV in CEF in varying degrees, and one siRNA targeting PA was best per-formed. The experimental results also showed that the inhibition effect was decreased with the time prolonged. This research provides a basis for further studying RNAi on AIV prevention and control.

Analysis of characteristics of whole-genome of influenza A H1N1 virus in Qingdao between year 2009 and 2011 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate characteristics of the whole-genome of influenza A H1N1 virus circulated in Qingdao from year 2009 to 2011.</p><p><b>METHODS</b>RNA of 35 influenza A H1N1 virus isolates circulated in Qingdao between year 2009 and 2011 was extracted and all segments were amplified by RT-PCR. The sequence was then detected and assembled by software Sequencher.25 HA full-length sequences published on GenBank were selected as reference. While MEGA 5.0 software package was explored for phylogenetic analysis to characterize the molecular feature with reference to the whole-genome sequence and the hemagglutinin (HA).1068 HA sequences of influenza A H1N1 virus isolated worldwide from August 2010 to March 2011 were downloaded for amino acid mutation analysis.</p><p><b>RESULTS</b>On the HA genes phylogenetic tree, the virus were separately divided into 4 clades in 2009-2010 and 2010-2011 surveillance season, each with a preponderant epidemic clade. The homogeneity of nucleotide and amino acids of HA isolates were 99.6%-99.9% and 99.1%-99.8% respectively in 2009-2010 surveillance season; 99.1%-99.6% and 98.2%-99.1% respectively in 2010-2011 surveillance season. The homogeneity of nucleotide and amino acids of the preponderant isolates were separately 98.8%-99.8% and 98.0%-99.6%. Compared with the vaccine strain, there were separately 14 and 12 variant amino acids of virus HA in the two surveillance season, involving 10 antigen sites and 5 positive selected sites. The sequence analysis of neuraminidase protein showed that the positions 247, 274 presented serine and histidine(S247, H274) respectively. The sequence analysis of M2 protein showed that the isolated A H1N1 viruses presented asparagine in amino acid site 31 (N31).</p><p><b>CONCLUSION</b>All the A H1N1 influenza virus circulated in Qingdao from year 2009 to 2011 presented continual variation and therefore caused antigenic drift. All the isolations were adamantane-resistance, but susceptible to inhibitors of neuraminidase.</p>