Two H5N1 avian influenza viruses (AIV), A/mallard/Huadong/S/2005 (S, IVPI = 2.65, in mallard) and A/mallard/Huadong/Y/2003 (Y, IVPI = 0, in mallard), were capable of distinct in pathogenicity to non-immunized mallards (Anas platyrhynchos). There were two amino acid residues difference in the HA cleavage site between two viruses, 322 (S, Leu; Y, Gln) and 329 (S, deletion; Y, Lys). Based on the variation, a series of recombinant viruses carrying HA gene either from S or Y virus with mutation at 322 and/or 329 were constructed via reverse genetics system to explore the influence of the two amino acid residues on viral pathogenicity in mallards. Recombinant viruses with S virus backbone were completely attenuated in terms of their virulence to ducks when position 322 (L322Q) and/or position 329 (-329K) of HA gene had been mutated. The critical role that L322 and -329 of HA protein from S virus play in the high virulence to ducks were influenced by the entire background of that protein because the recombinant virus with HA gene from Y and other seven genes from S were completely attenuated even if Q322L and K329- mutations of HA gene had been achieved. Recombinant viruses with Y virus backbone significantly increased their virulence to ducks when position 322 (Q322L) and/or position 329 (K329-) of HA gene had been mutated. All recombinant viruses carrying HA gene from Y with Q322L and/or K329-mutations and other seven genes from S were completely attenuated in terms of virulence to ducks whereas all recombinant viruses carrying HA gene from Y with same mutations and other seven genes from Y gained significant virulence. It seems that the compatibility among eight genes might be an important factor for HA to exert its functions. Results indicated that the mutation at amino acid position 322 and deletion at 329 in HA cleavage site significantly influence the pathogenicity of S and Y viruses in mallard, the compatibility among eight genes also contribute to the pathogenicity of both viruses in mallard.
Animals ; Birds ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; physiology ; Influenza A Virus, H5N1 Subtype ; genetics ; pathogenicity ; Structure-Activity Relationship ; Virulence

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.
Animals ; *Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus/chemistry/*genetics/metabolism ; Influenza A Virus, H9N2 Subtype/*genetics/metabolism ; Influenza in Birds/virology ; Phylogeny ; Poultry Diseases/*virology ; Sequence Analysis, RNA/veterinary

Since March 2009, pandemic A/H1N1/2009 influenza virus has been spreading throughout many countries including China. The emerged virus caused great harm to human health and social economy. Hemagglutinin (HA) is the most important viral surface glycoprotein, mainly possessing three kinds of functions: (1) binding to host cell receptor, (2) triggering the fusion between viral envelop and target cell membrane, (3) stimulating the body to generate the neutralizing antibody. Advances in the structure, primary function, evolution and antigenicity of pandemic A/H1N1/2009 influenza virus HA protein are reviewed in this paper.
Animals ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; pathogenicity ; physiology ; Influenza, Human ; epidemiology ; virology ; Pandemics

To isolate and identify the influenza virus in Shandong Province in 2009-2010 and analyze the genetic characteristics of hemagglutinin and neuraminidase gene, further study the variation of gene. A total of 17 126 nasopharyngeal swabs from fever patients were collected and detected by real time quantitative RT-PCR method. The results showed 4004 samples were pandemic influenza A (H1N1) virus positive, with an overall positive rate as 23.38%. The positive samples were incubated and cultured in MDCK cells. The HA and NA genes of isolated pandemic influenza A(H1N1) virus were sequenced, the homology analysis of the HA and NA genes showed an average of 96.9%-99.3% and 99.1%-99.6% sequence identity, respectively, compared with WHO-recommended vaccine strain. The genetic evolution and amino acid substitutions were performed with Mega 4.0 Software. Twenty one amino acids were changed in HA protein, of which 11 were located in the antigenic site; Sixteen amino acids were changed in NA protein, which didn't lead to the changes of enzyme sites. Furthermore, one glycosylation site of HA protein and NA protein were changed respectively. No H275Y mutation in NA protein was found. The results showed that the HA and NA genes of the epidemic strains were highly homologous, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process. All the strains were sensitive to oseltamivir.
China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Neuraminidase ; genetics ; Pandemics ; Phylogeny ; Time Factors

The molecular characterization and phylogenetic analysis of hemagglutinin (HA) genes of human influenza H3N2 viruses in Guangdong, China from 2007 to 2010 were studied in this study. By space-time sampling of strains, the HA genes of H3N2 strains from Guangdong were sequenced and searched from Internet, and then the variation and evolution of HA genes were conducted by Lasergene 7.1 and Mega 5.05 and evolutionary rates were analyzed by epidemiological data. The phylogenetic tree was established by alignment of 17 Guangdong strains and 26 global reference strains. Ks rates and Ka rates of HA genes were 2.06 x 10(-3)-2.23 x 10(-3) Nt/Year and 1.05 x 10(-3)-1.21 x 10(3) Nt/Year during 2007-2010, while the velocity of HA1 evolution of Ka was 3. 13 times than that of HA2 evolution. Compared with HA of vaccine strain A/Perth/16/2009, the genetic homologies of Guangdong strains in 2009 reached to 98.8%-99.7% and of Guangdong strains in 2010 reached to 98.0%-98.4%. There were some amino acid substitutions in five epitope regions of HA1 during 2007-2010, especially in B region (N160K) and D region (K174R/N); the K189E/N/Q and T228A in RBS (receptor-binding site) occurred in 2010 as two glycoproteins sites substituted impacted on the HA1 antigenicity. The antigenicity of epidemic H3N2 strains in 2010 was to some degree different that of the vaccine strain A/ Perth/16/2009. According to that there were variations of B and D epitopes and two sites of RBS and two glycoprotein in Guangdong H3N2 HA1 genes, WHO/ CDC should recommend new representative strains during 2011-2012 influenza seasons if H3N2 HA genes further evolve in the near future.
Amino Acid Substitution ; China ; Disulfides ; chemistry ; Epitopes ; genetics ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; Mutation ; Phylogeny

To study the genetic variation and evolutionary characteristics of H1N1 swine influenza virus, all the eight genes of LM were amplified by RT- PCR, cloned into pMD18-T vector and sequenced respectively. The results showed that neither insertion nor deletion was observed in nucleotides of LM. The amino acids sequence of cleavage site of HA is IPSIQSR decrease G, suggesting that LM did not have the molecular characteristics of high pathogen. HA had highly conservative N-glycosylation site at position 11, 23, 87 and 276 sites of HA1, and two more at position 154 and 213 sites of HA2. NA had highly conservative N-glycosylation site at position 58, 63, 68, 88, 146, and two more at position 44 and 235 sites, which might be one molecular characteristics of H1N1 subtype of SIV. The results of Bast showed HA gene had high homology to the strain of 'human-like' SIV (99%), while others had high homology to the 'classical' SIV. So it is inferred that HA of LM might originate from human-like linage swine influenza virus, while others might originate from 'classical' swine influenza virus.
Animals ; Cloning, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; Neuraminidase ; chemistry ; genetics ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction

Influenza virus is the causative agent of the seasonal and occasional pandemic flu. The current H1N1 influenza pandemic, announced by the WHO in June 2009, is highly contagious and responsible for global economic losses and fatalities. Although the H1N1 gene segments have three origins in terms of host species, the virus has been named swine-origin influenza virus (S-OIV) due to a predominant swine origin. 2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects. Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection. Primary sequence analysis of the 2009 S-OIV hemagglutinin (HA) reveals its closest relationship to that of the 1918 pandemic influenza virus, however, analysis at the structural level is necessary to critically assess the functional significance. In this report, we report the crystal structure of soluble hemagglutinin H1 (09H1) at 2.9 Å, illustrating that the 09H1 is very similar to the 1918 pandemic HA (18H1) in overall structure and the structural modules, including the five defined antiboby (Ab)-binding epitopes. Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV, and people born around the 1918 are resistant to the current pandemic, yet younger generations are more susceptible to the 2009 pandemic.
Animals ; Cloning, Molecular ; Crystallography, X-Ray ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; isolation & purification ; Influenza A Virus, H1N1 Subtype ; chemistry ; genetics ; immunology ; Models, Molecular ; Protein Conformation ; Swine ; virology

Antibodies, Viral ; blood ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; Humans ; Immunity, Cellular ; Immunity, Humoral ; Influenza A Virus, H1N1 Subtype ; immunology ; Influenza, Human ; epidemiology ; immunology ; Pandemics ; Time Factors

To demonstrate the phylogenetic evolution, the molecular characteristics of the motif of HA protein cleavage site and the varieties at the receptor binding sites of the hemagglutinin gene of the duck-origin H9N2 subtype avian influenza viruses, sequence alignment and phylogenetic analysis were performed by MEGA 4.1 Neighbor-Joining method.. The results revealed that the duck-origin H9N2 AIV viruses originated from CK/BJ/1/94-like and North-Ame-like, all the duck-origin H9N2 AIV viruses from mainland China belonged to CK/BJ/1/94-like and formed multiple genotypes through complicated re-assortment, while other duck-origin H9N2 AIV, isolated from other countries in Aisa, American and European such as Korea, Japan, Alberta, Austria, Switzerland, Iran, belonged to the North-Ame-like phylogenetic lineage. The amino acids at positions 183, 190, and 226 of the receptor binding sites of North-Ame-like group isolates had highly conserved H, E and Q respectively. In contrast with duck-origin H9N2 AIV viruses isolates from mainland China, the amino acids had N at positions 183, A, T, or V at 190, L or Q at 226, which was the same as the chicken-origin H9N2 AIV from mainland China. Most newly isolated chicken-origin H9N2 AIV in Fujian Province in Southern China had L at position 226 emphasized the higher risk of cross-infection between the chicken-origin and duck-origin H9N2 AIV in China.
Animals ; China ; Ducks ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; Influenza A Virus, H9N2 Subtype ; chemistry ; classification ; genetics ; isolation & purification ; Influenza A virus ; chemistry ; classification ; genetics ; Influenza in Birds ; virology ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Alignment

OBJECTIVETo analyse seasonal influenza epidemic situation in 2006, and to analyse the genetic and antigenic characteristics of viral hemagglutinin (HA) gene.

METHODSThe single-way hemagglutination inhibition (HI) tests were used to test the antigenic characteristics of these viruses from influenza surveillance network, and the HA1 genes were sequenced based on the antigenic test results according to different isolation times and sites.

RESULTSThe influenza virus types A and B co-circulated in 2006. influenza A H1N1 subtype and Victoria-like B influenza circulated preponderantly during this epidemic season. The HA1 gene sequence of H1N1 viruses showed that 192, 193, 196, 198 positions (located at antigenic site B) have an amino acid substitute, compared with the last circulating strain A/Hubeihongshan/53/2005(H1N1). Two amino acid changes at 142 and 144 positions compared with A/Yunnan/1145/2005 (H3N2). There was no change in influenza B viruses either Victoria-like B or Yamagata-like B virus, i.e . antigenic characteristics is analogous to B/shenzhen/155/2005 and B/tianjin/144/2005, respectively.

CONCLUSIONThe H1N1 and H3N2 influenza viruses had changing antigenic and genetic characteristics in 2006. Influenza virus types B did not change in 2006.

Amino Acids ; analysis ; China ; Hemagglutination Inhibition Tests ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; Influenza A Virus, H1N1 Subtype ; immunology ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; immunology ; isolation & purification ; Influenza B virus ; immunology ; isolation & purification ; Time Factors