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.
Animals ; Antibodies, Monoclonal ; Antibodies, Viral ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Hemagglutinins ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Molecular Docking Simulation

Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.
Animals ; Base Sequence ; Birds ; China/epidemiology* ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus/immunology* ; Hemagglutinins/genetics* ; Humans ; Influenza A Virus, H7N9 Subtype/isolation & purification* ; Influenza in Birds ; Influenza, Human/virology* ; Neuraminidase/genetics* ; Phylogeny ; RNA, Viral/genetics* ; Sequence Analysis, DNA

OBJECTIVEGene sequence data were clustered to explore evolution lineages of H3 antigen of influenza A virus.

METHODSAll data of H3 RNA sequence in NCBI Genbank and Influenza sequence database were downloaded and aligned in ClustalX while two step cluster method were applied to explore the data.

RESULTSAll sequences were aggregated into ten clusters, while seven of them mainly were human virus. Human virus and avian/other mammal virus were separated into different clusters distinctively, but coexisted into same clusters with swine virus. Time and host distribution were very distinctive in these clusters, but no geographic distribution features were found.

CONCLUSIONWith the interaction of human immunity system, H3 antigen mutated significantly every 5 - 7 years, and the speed of mutation had accelerated with the application of influenza vaccines in recent years. Mean while, human and swine influenza virus were not separated distinctly between clusters indicating that they had short inheritance distance. Result showed again that swine served as the mixer for antigenic recombination of different influenza virus.

Antigenic Variation ; genetics ; Antigens, Viral ; genetics ; Cluster Analysis ; Evolution, Molecular ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Hemagglutinins, Viral ; genetics ; immunology ; Humans ; Influenza A virus ; genetics ; immunology ; Mutation ; Sequence Analysis, DNA ; Viral Envelope Proteins ; genetics

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.
Actins ; genetics ; Animals ; Chick Embryo ; DNA Primers ; genetics ; Fibroblasts ; virology ; Hemagglutination ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Hemagglutinins ; genetics ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; growth & development ; physiology ; RNA Interference ; RNA Replicase ; genetics ; RNA, Small Interfering ; chemical synthesis ; genetics ; RNA-Binding Proteins ; genetics ; Real-Time Polymerase Chain Reaction ; Specific Pathogen-Free Organisms ; Transfection ; Viral Core Proteins ; genetics ; Viral Proteins ; genetics ; Virus Replication

OBJECTIVETo investigate the variations on hemagglutinin (H) gene of measles virus (MV) in Zhejiang province, and to analyze the differences with strains circulated both at home and abroad.

METHODSIn total, 33 MV strains isolated in Zhejiang province between 1999 and 2011 were collected.RNA of the isolated MV strains was extracted and the complete sequences on H gene were amplified using RT-PCR assay. The products were compared with the Chinese vaccine strain Shanghai-191, which was downloaded from GenBank, and other 95 different MV strains from all over the world.

RESULTS33 MV strains, isolated from the throat swab specimens collected from MV patients in Zhejiang province during 1999 to 2001, were used to conduct phylogenetic analysis with MV strains circulated in other areas of China during 1993 to 2007. The phylogenetic tree based on H gene sequences showed that all the Zhejiang MV strains located in H1a cluster, and no apparent time series and geographic restrictions were observed. Compared to the referenced vaccine strain Shanghai-191, the average variation rate on nucleotides and amino acids, and the evolutionary rate of H1a viruses from China during 2003 to 2011 were separately 5.15%, 4.44% and 5.81%, which were higher than the rates of H1a viruses during 1965 to 1993 (4.75%, 3.86% and 5.30%), and the rates of viruses during 1994 to 2002 (4.80%, 4.08% and 5.37%).However, the dn/ds ratios of strains within the three time periods were 0.19,0.21 and 0.23 respectively, which indicated that no evidence of positive selection was found on H1a MV strains during 1993 to 2011. A 24 stable amino acid variation sites on H gene was found between H1a viruses during 2003 to 2011 and the vaccine strain Shanghai-191. The largest variation occurred between vaccine and H1a strains, with 0.053 of the p-distance and 26-28 of amino acid mutations.However, only 15 stable amino acid variations were found between vaccine strain and genotype B3 or D4 strains.In addition, significant differences were found between H1a viruses and genotype B or D viruses, with 0.074 and 0.071 of p-distance and 27-33 of amino acid differences.

CONCLUSIONSignificant differences were found on H gene between MV strains subtype H1a and vaccine strains and other genotype strains. The variations were enlarged with the time coursing; therefore, the surveillance on variation of Chinese MV strains should be taken into account.

China ; epidemiology ; Genotype ; Hemagglutinins, Viral ; genetics ; Humans ; Measles ; epidemiology ; virology ; Measles virus ; classification ; genetics ; isolation & purification

Improving expression of antigen is critical to the immunogenicity of DNA vaccines. To achieve this goal, we modified the NDV F48E9 strain HN gene by optimizing the condon usage and inserting the secretary leader sequence [A/Goose/Guangdong/1/96 (H5N1) HA gene, Accession No. AF144305]. The HN gene modified and knocked the signal peptide off were named SoptiHN and optiHN. The three sequence: SoptiHN, optiHN and the NDV F48E9 strain HN gene were inserted into the vector pVAX1 and vector pVAX1-CpG including CpG-ODN sequence respectively. Then we got six recombinant plasmids: pV-SoptiHN, pVC-SoptiHN, pV-optiHN, pVC-optiHN, pV-HN and pVC-HN. By optimizing condon usage in transiently transfected 293T cells, expression levels of HN gene were higher from the codon-optimized gene than the counterpart. Moreover, both optimization of condon usage and addition of signal peptide could improve expression of HN gene in vitro.
Animals ; Chickens ; Codon ; HN Protein ; genetics ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; Newcastle Disease ; immunology ; prevention & control ; Newcastle disease virus ; classification ; genetics ; Vaccines, DNA ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

Vaccination is one of the most effective and cost-benefit interventions that prevent the mortality and reduce morbidity from infectious pathogens. However, the licensed influenza vaccine induces strain-specific immunity and must be updated annually based on predicted strains that will circulate in the upcoming season. Influenza virus still causes significant health problems worldwide due to the low vaccine efficacy from unexpected outbreaks of next epidemic strains or the emergence of pandemic viruses. Current influenza vaccines are based on immunity to the hemagglutinin antigen that is highly variable among different influenza viruses circulating in humans and animals. Several scientific advances have been endeavored to develop universal vaccines that will induce broad protection. Universal vaccines have been focused on regions of viral proteins that are highly conserved across different virus subtypes. The strategies of universal vaccines include the matrix 2 protein, the hemagglutinin HA2 stalk domain, and T cell-based multivalent antigens. Supplemented and/or adjuvanted vaccination in combination with universal target antigenic vaccines would have much promise. This review summarizes encouraging scientific advances in the field with a focus on novel vaccine designs.
Animals ; Disease Outbreaks ; Hemagglutinins ; Humans ; Influenza Vaccines ; Influenza, Human* ; Mortality ; Orthomyxoviridae* ; Pandemics ; Seasons ; Vaccination ; Vaccines* ; Viral Proteins ; Viruses

This study aims to investigate the genetic characteristics of hemagglutinin in wild-type measles viruses in Henan Province, China and to provide a basis for measles control and elimination. Specimens were collected from suspected measles cases in Henan during 2008-2012. Cell culture was performed for virus isolation, and RT-PCR was used to amplify hemagglutinin gene. The PCR products were sequenced and analyzed, including construction of phylogenetic tree and analysis of the distance between the isolated virus and the reference virus; then, the variations in predicted amino acids were analyzed. The results showed that 12 measles viruses were isolated in Henan Province and identified as H1a genotype; the nucleotide and amino acid homologies were 98.0%-100% and 97.2%-99.8%, respectively. One glycosylation site changed in all the 12 sequences because of the amino acid mutation from serine to asparagine at the 240th site, as compared with Edmonston-wt. USA/54/A. Overall, the wild-type measles virus genotype circulating in Henan Province from 2008 to 2012 was H1a, with high homology between strains; there were some variations in amino acid sequences, resulting in glycosylation site deletion.
China ; Hemagglutinins ; genetics ; Humans ; Measles ; virology ; Measles virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

We produced high pathogenic avian influenza H5N1 haemagglutinin protein HA1 in recombinant Pichia pastoris in a 10 L fermentor, to establish a high-density cell fermentation method. We studied the effects of different factors such as culture temperature, induced temperature, methanol feeding methods, trace elements on the growth of Pichia pastoris, the yield and the biologic activity of recombinant HA1 protein. The culture temperature in pre-induced and induced stage were optimized at 25 degrees C to adapt cell growth and recombinant protein expression, and induced temperature at 25 degrees C also resulted in higher biologic activity of rHA1 than at 30 degrees C. The binding activity of rHA1 against a wide-spectrum neutralizing antibody was susceptible to the presence of any trace elements, although trace elements would essentially benefit for the cell fermentation. As a conclusion, the expression level of rHA1 produced with optimized fermentation process reached 120 mg/L, which was 10.5 times higher than the one produced in regular shaking flask. The resultant high-density cell fermentation can likely produce rHA1 of H5N1 in large scale.
Fermentation ; Hemagglutinins, Viral ; biosynthesis ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.
Animals ; Cattle ; Dogs ; Hemagglutinins, Viral ; chemistry ; metabolism ; Influenzavirus C ; physiology ; Orthomyxoviridae Infections ; metabolism ; virology ; Protein Conformation ; Protein Folding ; Protein Processing, Post-Translational ; Viral Fusion Proteins ; chemistry ; metabolism