Glycosylation at 11Asn on hemagglutinin of H5N1 influenza virus contributes to its biological characteristics.

A stem glycosylation site of hemagglutinin (HA) is important to the stability of the HA trimmer. A previous study shows that the stem 10/11 overlap glycosylation site of the H5 subtype avian influenza virus may influence the cleavage of HA, whereas the exact site and its effect on virulence remain unclear. In this study, site-directed mutagenesis was used to generate single or double mutant rSY-Δ10(10NNAT), rSY-Δ11(10NNSA), and rSY-Δ10/11(10NNAA) of the overlapping glycosylation site (10NNST) on the HA of A/Mallard/Huadong/S/2005(SY). By using Western blot analysis, we show that both rSY-Δ11 and rSY-Δ10/11 mutant viruses had significant delay on HA cleavage and a reduced HA molecular mass compared to the wild-type virus rSY, while the rSY-Δ10 mutant virus exhibited a similar HA molecular mass to that of the wild-type virus rSY. Interestingly, both rSY-Δ11 and rSY-Δ10/11 mutant viruses reverted their glycosylation sites at 11N after passage, indicating that 11N is a true and critical glycosylation site. Compared to the wild-type virus rSY, rSY-Δ11 and rSY-Δ10/11 mutant viruses had decreased growth rates, reduced thermo- and pH-stability, decreased pathogenicity, and limited systemic spread. Therefore, our study suggests that the 11N glycosylation site plays a key role in HA cleavage, structural stability and pathogenicity in H5 subtype avian influenza virus.

Design of "Off-On-Off" fluorescence sensors for Heparin detection by precise modulation of molecular structure.

In this strategy, the fluorescence sensor Nap-Co-T1 employing the fluorescence resonance energy transfer (FRET) mechanism was designed and synthesized to have an efficient response to Heparin, and the FRET mechanism was explored for different excitation-emission wavelengths with different distances between the energy acceptor and the energy donor (comparing with fluorescence sensor Nap-TPA-T2). Upon the addition of Heparin, the fluorescence emission of Nap-Co-T1 was turned on at 565 nm, and the fluorescence color changed of the solution from colorless to bright yellow. The limit of detection (LOD) was as low as 0.04 µg/mL. With the addition of antagonistic protamine (PRTM) to the sensor complex with Heparin, the fluorescence emission was turned off to a certain extent, and the reversibility of the "off-on-off" system was maintained for five cycles or more. In addition, Nap-Co-T1 provides rapid and sensitive detection of Heparin in human serum albumin solution and artificial urine and is highly sensitive to environmental viscosity.

Enhanced cross-lineage protection induced by recombinant H9N2 avian influenza virus inactivated vaccine.

BACKGROUND: Antigenic drift of H9N2 low pathogenic avian influenza viruses (AIV) may result in vaccination failure in the poultry industry and thus a cross-protective vaccine against H9N2 AIV is highly desirable. METHODS: A series of H9N2 recombinant viruses with the internal genes of A/Puerto Rico/8/34 (H1N1, PR8) were generated, based on the compatibility between HA and NA, the effect of HA deglycosylation, and protective antigenic epitopes in HA. After evaluation of their biological and immunological characteristics, three recombinant AIVs with the internal genes of the Y280-like strain SN were selected for protective efficacy studies. RESULTS: The recombinant viruses rHASNNA3, rHASN-△200, rHASN-△287, and rHASN-R92G-E93K displayed good cross reactivity and induced higher neutralization antibody titers against both SN and the F98-like strain YZ4. Furthermore, those recombinant viruses had a higher EID50 in chicken embryos after the replacement of internal-gene backbone from PR8 to SN. The rSNHA-△200 induced better protection in immunized chickens against challenge of homologous and heterologous H9N2 avian influenza viruses when compared with the wild type strain. CONCLUSION: The recombinant virus rSNHA-△200 can be used as a potential broad-spectrum vaccine against H9N2 avian influenza.