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In March 2012, an H7N7 subtype avian influenza virus (AIV) named A/wild goose/Dongting/PC0360/2012 (H7N7) (DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization.RNA was extracted from environment samples (including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed.Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in Mainland China in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus.Strengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

Five H9N2 avian influenza virus strains were isolated from the environmental samples in live poultry market in Qinghai Lake region from July to September, 2012. To evaluate the phylogenetic characteristics of these H9N2 isolates, the eight gene segments were amplified by RT-PCR and sequenced. The phylogenetic and molecular characteristics of the five strains were analyzed. The results showed that the HA genes of five strains shared 93. 2%-99. 1% nucleotide identities with each other, and the NA genes shared 94. 5%-99. 8% nucleotide identities. The HA cleavage site sequence of the A/environment/qinghai/ 017/2012 isolate was PSKSSRGLF, and the HA cleavage site sequences of the other four strains were all PSRSSRGLF. The HA receptor-binding site had the Q226L mutation. The M1 gene segment had the N30D and T215A mutations. The phylogenetic analysis showed that the five strains were similar to the virus A/chicken/Hunan/5260/2005 (H9N2) isolated in Hunan Province, China and were reassortant genotype viruses; the HA, NA, and NS genes belonged to the Y280-like lineage; the MP gene belonged to the G1-like lineage; the NP, PB1, PB2, and PA genes belonged to the F98-like lineage.

To study the prevalence and variation of influenza A(H3N2) viruses, the antigenic and genetic characteristics of influenza A(H3N2) viruses circulating in Mainland China during April 2011 to March 2012 were analyzed. The results showed that influenza A(H3N2) viruses increased gradually since 2012 and became the dominant strain since March. The viruses were antigenically closely related to the vaccine strain A/PER/16/09 (87.2%) and the representative virus A/FJ/196/09 (76.0%) in Mainland China. The genetic characteristics analysis results showed that recently isolated viruses belonged to the Vic/208 clade, and most of the low reaction strains also fell into the same clade. Crystal structure analysis of HA protein found that, compared with the vaccine strain A/PER/16/09, the recently isolated viruses had amino acid substitutions in the antigenic site A, B and C areas, in addition to gaining potential glycosylation sites at the amino acid position of 45 of HA and 367 of NA. Although the majority of circulating influenza A (H3N2) viruses in 2011-2012 season in Mainland China were antigeniclly matched by current influenza vaccine strain and the selected representative viruses, low reaction strains have increased since 2012, therefore it is necessary to strengthen the surveillance on the variation of influenza virus and to provide solid information for the vaccine strain selection.

To analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market (LPM) in 2009 in Guangdong province of China.PCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed.The genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus.A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.

Pdm09 virus outbreak occurred in Mainland China in May 2009, a few months later, the prevalence of seasonal H1N1(sH1N1) influenza virus that already circulated in human for tens of years began to decline and disappeared afterwards. To identify the reason for the rapid decline of sH1N1 in mainland China, we sequenced the HA1 of sH1N1 during 2006-2011, and then analyzed the selective pressure in different phases. Our results showed before Pdm09 outbreak, the omega value was 0. 36 while after Pdm09 outbreak the omega value was 0. 28 and significant difference (t test, P0. 05) was identified. We concluded that sH1N1 obtained stronger purifying selection after Pdm09 outbreak in China. This might one of the major reasons causing the disappearance of sH1N1 in human.

In order to understand the prevalence and variation of influenza B viruses, the antigenic and genetic characteristics of influenza B viruses circulating in Mainland China during April, 2011 to March, 2012 were analyzed. The results showed the B Victoria lineage viruses were much more prevalent than B Yamagata lineage during this period, phylogenetic analysis showed vast majority of Victoria lineage viruses belong to genetic group 1, intra-clade reassortant between HA1 and NA gene was identified in a minor proportion of the viruses. 72.8% of the B/Victoria-lineage viruses were antigenically closely related to the vaccine strain B/Brisbane/60/2008. B Yamagata component was not included in the trivalent influenza vaccine in China during the study period, however vast majority of B Yamagata lineage viruses were antigenically and genetically closely related to the representative virus B/Hubei-Wujiagang/158/2009(97.8%) and B/Sichuan-Anyue/139/2011(85.2%) in China, reassortant between HA1 and NA was not identified in B Yamagata lineage viruses. Overall, the predominant circulating influenza B viruses in 2011-2012 season in China were matched by current influenza vaccine and the selected representative viruses were proved to represent the antigenic and genetic characteristics of the circulating viruses.

To conduct a full genome sequence analysis for genetic characterization of an H3N8 influenza virus isolated from drinking water of a domestic duck farm in Poyang Lake area in 2011.The virus was cultivated by specific pathogen free (SPF) chicken embryo eggs and was subtyped into hemagglutinin (HA) and neuraminidase (NA) by real-time PCR method. Eight gene segments were sequenced and phylogenetic analysis was conducted.The NA gene of this virus belongs to North American lineage; other seven genes belong to Eurasian lineage. Compared with the viruses containing NA gene, the PB2 and PB1 gene came from different clades. And this indicates that the virus was a novel reassortant genotype. The HA receptor binding preference was avian-like and the cleavage site sequence showed a low pathogenic feature. There was no drug resistance mutation of M2 protein. The mutations of Asn30Asp, and Thr215Ala of the M1 protein implied the potential of pathogenicity increase in mice.The finding of novel genotype of H3N8 virus in drinking water in this duck farm near Poyang Lake highlighted the importance of strengthening the surveillance of avian influenza in this region, which could contribute to pinpointing the influenza ecological relations among avian, swine, and human.

To understand the possible origins, genetic re-assortment and molecular characterization of 4 highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province, between 2006 and 2009.H5N1 PCR test-positive specimens were inoculated in embryonated eggs while H5N1 virus was isolated and genomes sequenced. Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 4.0.All gene segments of the 4 viruses were avian in origin. No re-assortment was found between avian influenza A (H5N1) viruses and human seasonal influenza viruses. Viruses that isolated from domestic poultry shared high similarity with the 4 human viruses in gene homology. Data from the whole genome phylogenetic analysis showed that the 4 viruses were in clade 2.3.4, while 2 viruses belonged to genotype V, and another 2 were new genotypes. Results from molecular characterization showed that amino acid sequences of HA cleavage site of the 4 viruses were PLRERRKR/G. All 4 viruses had A160T mutation in HA, a 20 amino acid deletion in the neuraminidase (NA) stalk at position 49 - 68, and a 5 amino acid deletion in the non-structural protein 1 (NS1). Most sites in the HA molecules showed that the viruses preferentially bound to avian influenza virus receptor. However, T192I mutation that might enhance the α 2, 6-linked sialic acid human influenza receptor binding had emerged in HN/1/09 and HN/2/09. D701N mutation of PB2 that increased the virulence in mice was found in HN/1/08. Analysis on drug resistance gene amino acid showed that all 4 viruses were sensitive to amantadine and oseltamivir.Highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province from 2006 to 2009 were avian in origin, and the 4 viruses belonged to different genotypes. Some mutations that related to virulence and receptor binding positions had emerged in some of the strains.

To under stand influenza A viruses epidemic, antigenicity and genetic characteristics variation between the vaccine and Circulation strains during 2004-2008 year in China.The influenza A viruses (H1N1, H3N2) isolated from 2004-2008 year were under took antigenic and sequence analysis. Influenza A virus antigenicity and genetic characteristics were analyzed thought amino acid variation compassion of HA1 protein of influenza A virus isolates.The antigenicity of influenza H1N1 subtype viruses isolated from 2004 to 2007 is very similar with vaccine strain A/New Caledonia/20/1999 (HIN1)-like virus. The influenza H1N1 viruses circulated in 2008 year had similar antigenic characteristics with A/Brisben/59/2007 (H1N1) which is component of influenza vaccines for northern hemisphere 2008-2009 year. The influenza H3N2 subtype viruses of 2004-2005 year had antigenic variation comparatively with vaccine strain A/Fujian/411/12002 (H3N2), The antigenicity of 2006-2007 H3N2 viruses and 2008 year's is A/Wiscansin/67/2006(H3N2) and A/ Brisben/10/2006(H3N2) respectively.There is change of influenza A viruses (H1N1, H3N2) antigenic and genetic characteristics during 2004-2008 in China.

The NA genes of 395 strains of human H3N2 influenza virus isolated from 1996 to 2005 in China were sequenced, analyzed with bioinformatics tools. The NA nucleotide sequence of phylogenetic tree showed a main evolution branch with multiple short side branches. The strains in the same year may be divided into several branches. There was an obvious lag between vaccine strains recommended by WHO and the Chinese circulating strains in phylogenetic tree of the NA nucleotide. The result also showed no amino acid deletion and insertion in the NA. In NA antigen sites, where including residues 197-199 aa, 431-434 aa and 339-347aa the mutation was higher, in contrast, the residues including 153 aa, 328-336 aa, 367-370aa and 400-403 aa, the mutation was lower. Besides the antigenic determinant sites, there also had the other amino acid mutated highly, such as 18, 23, 30, 93, 143, 208, 216, 221, 249, 265, 267, 307, 385 and 437 aa, among them 143 and 267 mutation were higher than that in antigenic determinant sites, their biological significance are not clear yet. The neuraminidase active-site residues in NA were highly conservative and the same were the disulphide bond and the glycosylation sites in NA. In conclusion, our analysis provides some information for influenza prevention and control and the NA inhibitor medicine application.

To understand the evolutionary characterization of HA1 of H1N1 influenza virus HA gene circulaing from 1981 to 2005 in China, viral RNAs of 370 H1N1 strains were extracted and transcribed into cDNA by reverse transcriptase and amplified by PCR. The products of PCR were sequenced. The sequences were analyzed through biometic software. The results showed that all the four antigenic sites were mutated, bigger change occurred on the Sb and Ca sites; the 130 loop of receptor binding sites(RBS) of HA1 amino acid deleted at the 134th site in 1991 firstly, then the number of the deleted strains were increasing, since 2000, all the strains had deleted at the 134th site, and simultaneously, the amino acid at 137th site was substituted by S for T. The change of HA1 glycosylation sites was found and 7 sites kept stable from 2000 to 2005. The H1N1 strains of the same year almost clustered in the same group on the phylogenetic tree and were irrelevant to virus isolated time and area. There appeared two groups of 2005 H1N1 virus strains that differed in time of virus isolation.

NS1 gene was amplified from an H5N1 influenza virus, A/Anhui/01/2005 for cloning, sequence analysis and later expression in Escherichia coli. The result showed that NS1 of A/Anhui/01/2005 strain had the close phylogenetic relationship with that of H5N1 avian influenza strains recently isolated in Fujian and Hunan. Correspondingly, its amino acid sequence showed the highest homology with that of Fujian and Hunan strains. The amino acid position of 92 involved in virus virulence was Asp, contrast to Glu in A/HK/156/97. Five amino acid deletion from 80 to 84 was also found in A/Anhui/01/2005, which was considered as a contributor to virus resistance against cytokine, such as IFN, TNF, etc. A motif binding to CBSF, conveted to GFWEN, which was different from previous GLEWN found in other 19 strains. Besides, the PL motif of ESEV, binding to PDZ domain, is the same as previous high-mortality H5N1 isolates. Furthermore, this NS1 was efficiently expressed in Escherichia coli and the highly purified product demonstrated wonderful activity as confirmed by Western blot. As a result, the work paves the way for further understanding the role of NSI in human H5N1 infection and development of new antiviral drugs against influenza virus.

To analyze the genetic and antigenic characteristics of hemagglutinin (HA) gene of human influenza B virus isolated from the mainland of China since 2004-2005.The single-way hemagglutinin inhibition (HI) tests were used to test the antigenic characteristics, and the HA1 gene was sequenced based on the antigenic results.The Yamagata-like and Victoria-like viruses co-circulated in 2004-2005. For the Yamagata-like virus, the single-way HI results showed that 3.7% and 4.5% of the viruses had 4-fold greater HI titer difference compared with B/Shanghai/361/02 in 2004 and 2005, respectively. The HA1 sequence data showed that the virus had amino acid mutation, and there was one more glycosylation site at 196th site. For the Victoria-like virus, the single-way HI results showed that 8.5% and 20.6% of the viruses had 4-fold greater HI titer difference compared with B/Hong kong/330/01 in 2004 and 2005, respectively. The HA1 sequence data showed that the virus had replacement of 9 amino acids, and there was one more glycosylation site at 197th site.The results showed that influenza B viruses had changed antigenic and genetic characteristics compared with B/Shanghai/361/02, B/Hong kong/330/01 in 2004-2005.

To analyze the genetic and antigenic characteristics of hemagglutinin (HA) gene of human influenza (H1N1) virus isolated from the mainland of China since 2004 to 2005.The single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics, and the HA1 gene was sequenced based on the antigenic results.The single-way HI results showed that no virus isolates had 4-folds greater HI titer compared with A/Shanghai/1/1999 (H1N1) in 2004, but there was 6.3% virus had 4-fold greater difference in 2005. The HA1 sequence data showed that the H1N1 virus had the following amino acid mutations such as 54 KR, 90 TK, 101 YH, 149 RK, 169 VA, 190 DN, 212 RK, 219 KR, 245 WR, 246 YF, 258 TN, 318 VA and the 54 and 190 amino acids located in antigenic group of HA1.The H1N1 virus was changing in antigenic and genetic characteristics.

The Victoria lineage represented by B/Victoria/2/87 is one of the two major distinctive haemagglutinin (HA) lineages of influenza B virus, and its recent re-emergence has aroused great concerns. However, it remains unknown when, where, and how this HA lineage emerged in the world. In this study, the HA1 domain of the HA gene of fourteen influenza B viruses isolated in China in 1972-1984 was sequenced. The sequences were phylogenetically analyzed with the HA1 sequences of 41 other important influenza B isolates. The results unveiled some earlier footprints of the Victoria lineage in China, and the epidemic history of the Victoria lineage could be traced back from the year 1985 to 1975. Moreover, phylogenetic analysis, the history of China, and the epidemiology of influenza B virus indicated that the Victoria lineage possibly emerged in China in the 1970s through gradual evolution from a minor lineage.

To study the antigenic and genetic characteristics of influenza (H3N2) virus circulated in China in 2004.Single-way and cross-way hemagglutination inhibition (HI) tests were firstly used to determine the reactivity with the reference serum of virus isolates. Based on the serological results, virus isolates were selected according to the different time and location in China in 2004. The HA1 domain of HA gene of those virus isolates were then sequenced in order to analyze the gene characterization.Single-way HI test results showed that 52.3% of isolates showed 4 folds or more HI titer difference compared to A/Fujian/411/2002 (H3N2) itself (international reference strain in 2004). Cross-way HI test results showed that the antigenic ratio was 4. The nucleic acid and amino acid sequence data of HA1 domain showed that the mutated virus appeared in early February of 2004, and became the dominant circulating strain gradually. There were four important mutant positions, they were 159 YF, 189 SN, 145 KN, 226 VI, respectively. The results also indicated that the mutated viruses originated from southern China, then transmitted to northern China, according to the analysis of time and location distribution.The HA1 domain of HA gene of influenza virus (H3N2) isolated from 2004 in China showed mutation and antigenic drift, and the mutated viruses were becoming the dominant circulating strain in China, and showed amino acid sequence difference compared to A/Fujian/411/2002 (H3N2) A/Wellington/1/2004 (H3N2), the vaccine components pronounced by WHO for 2004-2005 northern hemisphere and 2005 southern hemisphere respectively, which suggested that further surveillance should be conducted to monitor the virus mutation in circulation.

To understand whether pigs play a role in human infection with avian influenza A H9N2 viruses.The target gene was amplified by RT-PCR, and the PCR product was linked to PGEM-T Vector (Promega, USA) at 4 degrees C, the recombined plasmid was transferred into dH5a bacteria, and the positive colonies were selected and identified with restriction endonuclease. Afterwards, they were sent to Liu He Tong Company in Beijing for nucleotide sequencing. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseq (Version 3.69) software.The genomic characterizations of A/Swine/Shandong /5/2002(H9N2) and A/Swine/Shandong/10/2002(H9N2) viruses were different from those of H9N2 viruses which were isolated either from men or from chickens. The genomic characteristics of H9N2 viruses isolated from humans in China mainland were similar to those of H9N2 viruses isolated from chickens. Whereas, the genomes of H9N2 viruses isolated from men in Hong Kong, China were closely related to those of H9N2 viruses isolated from quails. Avian influenza A H9N2 viruses not only have wide range of host, but their genomes are also diverse.Avian influenza A H9N2 viruses can directly infect human. Avian influenza A H9N2 viruses did not require to pass through the pigs as mixing vessels prior to infecting man.