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.
Animals ; Antibodies, Viral ; Birds ; Hemagglutinin Glycoproteins, Influenza Virus ; Influenza A Virus, H5N1 Subtype ; Influenza Vaccines ; Influenza in Birds ; Pichia ; Vaccination

The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.
Animals ; China ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; pathogenicity ; Influenza in Birds ; virology ; Mice, Inbred BALB C ; Phylogeny ; Poultry

During 2006, H5N1 HPAI caused an epizootic in wild birds, resulting in a die-off of Laridae in the Novosibirsk region at Chany Lake. In the present study, we infected common gulls (Larus canus) with a high dose of the H5N1 HPAI virus isolated from a common gull to determine if severe disease could be induced over the 28 day experimental period. Moderate clinical signs including diarrhea, conjunctivitis, respiratory distress and neurological signs were observed in virus-inoculated birds, and 50% died. The most common microscopic lesions observed were necrosis of the pancreas, mild encephalitis, mild myocarditis, liver parenchymal hemorrhages, lymphocytic hepatitis, parabronchi lumen hemorrhages and interstitial pneumonia. High viral titers were shed from the oropharyngeal route and virus was still detected in one bird at 25 days after infection. In the cloaca, the virus was detected sporadically in lower titers. The virus was transmitted to direct contact gulls. Thus, infected gulls can pose a significant risk of H5N1 HPAIV transmission to other wild migratory waterfowl and pose a risk to more susceptible poultry species. These findings have important implications regarding the mode of transmission and potential risks of H5N1 HPAI spread by gulls.
Animals ; Birds ; Charadriiformes* ; Cloaca ; Conjunctivitis ; Diarrhea ; Encephalitis ; Hemorrhage ; Hepatitis ; Influenza A Virus, H5N1 Subtype* ; Lakes ; Liver ; Lung Diseases, Interstitial ; Myocarditis ; Necrosis ; Pancreas ; Pathology* ; Poultry ; Virulence

The occasional influenza pandemics and the seasonal influenza epidemics have destroyed millions of lives since the last century. It is therefore necessary to understand the virus replication patterns as this provides essential information on the virus infectivity, pathogenicity and spread patterns. This study aimed to investigate the replication of avian influenza A virus H5N1 (A/Chicken/Malaysia/5858/2004) in MDCK cells. In this study, the TCID50 (50% tissue culture infectious dose) of AIV H5N1 was first determined. The MDCK cells were then infected with AIV H5N1 at TCID50 for 0-48 h. The CPE (cytopathic effect) was observed and cell death was determined hourly. The virus-infected cells and media were subsequently collected for gene analysis. The results showed that the TCID50 of AIV H5N1 was 10-9 dilution. The CPE percentage showed a strong and positive correlation with the infection period (r = 1.0, n = 9, p < 0.01). The amount of a highly conserved influenza viral gene, M2 gene amplified from infected media (r = 0.471, n = 9, p= > 0.05) and infected cell (r = 0.73, n = 9, p < 0.05) were also positively correlated with the infection period. In conclusion, although CPE started to be observed in the early time points of infection, however, the M2 gene was only amplified from the infected media and cells after 48 h and 24 h, respectively. This signifies that AIV H5N1 used in this study is pathogenic and it is able to cause severe cytopathology to host cells even at low virus load.
Influenza, Human ; Influenza A Virus, H5N1 Subtype

OBJECTIVETo study the inhibitory activities of 3-O-β-chacotriosyl benzyl ursolate and its derivatives as potential new anti-influenza virus agents against the entry of H5N1 influenza viruses into the target cells.

METHODSFour target compounds were designed and synthesized, which were structurally related to the lead compound 3-O-β-chacotriosyl methyl ursolate (1). The inhibitory activities of these compounds were tested at a cellular level psuedovirus system targeting H5N1 influenza viruse entry.

RESULTS AND CONCLUSIONThe compounds 1b, 1c and 1d showed potent inhibitory activities against the entry of A/Thailand/Kan353/2004 pseudovirus into the target cells, and among them compound 1d showed the strongest inhibitory activity with an IC50 value of 0.96 ± 0.10 µmol/L. The structure-activity relationship analysis of these compounds indicated that when 17-COOH of ursolic acid was esterified, introduction of Me groups rather than aryl groups more strongly enhanced the inhibitory activity. Changing 17-COOH of ursolic acid into amide could increase the antiviral activity and decrease the cytotoxicity of the compounds in MDCK cells.

Animals ; Antiviral Agents ; chemistry ; Dogs ; Influenza A Virus, H5N1 Subtype ; drug effects ; physiology ; Madin Darby Canine Kidney Cells ; Structure-Activity Relationship ; Triterpenes ; chemistry ; Virus Internalization ; drug effects

To set up a new rapid method for the rapid determination of influenza virus H5N1 and H7N9 basing on the Multi-Analyte Suspension Array (MASA) technology. Sequence analysis and design of degenerate primers and specific probes were set in the comparison and analysis of H5, N1, H7 and N9 genes. In combination with MASA technology, these primers and probes were used for the determination of samples of H5N1 and H7N9 and other subtypes ( H1N1, PH1N1, H5N2, H3N2 and H9N2). We developed a rapid determination method. This method had high specificity and sensitivity that could detect H5N1 and H7N9 at one time, and could detect samples that containing 10 copies of H5N1 and H7N9. This determination method could be used for rapid determination of influenza virus H5N1 and H7N9 at one time.
Humans ; Influenza A Virus, H5N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H7N9 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Oligonucleotide Array Sequence Analysis ; methods

This study aims to investigate the virological impact of the stalk region and cysteine (C) in neuraminidase (NA) of influenza A/Anhui/1/05 (H5N1) and A/Ohio/07/2009 (H1N1) viruses. The NA of A/ Anhui/1/05 (H5N1), defined as AH N1, lacked 20 amino acids (including C, defined as s20) as compared with NA of A/Ohio/07/2009 (H1N1) (defined as 09N1). We deleted s20 of 09N1 to construct 09N1-s20, and inserted s20 into AH N1 to construct AH N1+s20. To investigate the impact of C on the biological function of NA, we deleted C in 09N1 to construct 09N1-C and inserted C into AH N1 to construct AH N1-C. The pseudo-type viral particle (pp) system was used to evaluate the impact of these mutants on virology. The combination of 09N1-C and 09H1 (defined as 09H1::09N1-C) showed an infectivity 8 times that of the wild type 09H1::09N1, while the infectivity of the combination of AH N1+C and AH H5 (defined as AH H5::AH N1+C) was much lower than that of the wild type AH H5::AH N1. The infectivity of the combination of 09N1-s20 and 09H1 (defined as 09H1::09N1-s20) was 4 times that of the wild type 09H1::09N1; the infectivity of the combination of AH N1+s20 and AH H5 (defined as AH H5:: AH N1+s20) was 1/7 that of the wild type AH H5::AH N1. The co-existence of 09N1-C and AH H5 displayed 6 times the infectivity of AH H5::09N1, while the infectivity of 09H1::AH N1+C was very low. Multimer analysis showed that in the wild type 09N1, the forms of NA were dimer > tetramer > monomer; the major component of NA in 09N1-C was monomer; in 09N1-s20, the forms of NA were monomer > dimer. AH N1 was mainly composed of monomer; in AH N1+s20, the forms of NA were dimer > monomer > tetramer; in AH N1+C, the forms of NA were dimer > tetramer. Deletion of C or s20 from 09N1 did not change the expression of NA. The study suggested that deletion of C from the stalk region of NA in A/Ohio/07/2009 (H1N1) increases infectivity. Insertion of C into NA's stalk region of A/ Anhui/1/05 (H5N1) significantly decreases infectivity. Cysteine deletion in the stalk region is important for the infectivity of A/Anhui/1/05 (H5N1) and A/Ohio/07/2009 (H1N1). It may interfere with the infectivity via changes in NA polymerization.
Amino Acid Motifs ; Humans ; Influenza A Virus, H1N1 Subtype ; chemistry ; enzymology ; genetics ; pathogenicity ; Influenza A Virus, H5N1 Subtype ; chemistry ; enzymology ; genetics ; pathogenicity ; Influenza, Human ; virology ; Neuraminidase ; chemistry ; genetics ; metabolism ; Viral Proteins ; chemistry ; genetics ; metabolism ; Virulence

Animals ; Humans ; Influenza A Virus, H5N1 Subtype ; immunology ; pathogenicity ; Poultry ; virology

OBJECTIVETo prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China.

METHODSRecombinant 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.

RESULTSThe 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics.

CONCLUSIONThe 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.

Animals ; Chick Embryo ; Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; prevention & control ; virology ; Neuraminidase ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, Synthetic ; immunology

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.
Amino Acid Sequence ; Animals ; Female ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Interferon-gamma ; biosynthesis ; Lung ; pathology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data