This study demonstrated the terminated sialo-sugar chains (Neu5Acα2,6Gal and Neu5Acα2,3Gal) mediated specificity enhancement of influenza virus and chicken red blood cell (RBC) by hemagglutination assay. These glycan chains were immobilized on the gold nanoparticle (GNP) to withhold the higher numbers. With the preliminary optimization, a clear button formation with 0.5% RBC was visualized. On the other hand, intact B/Tokio/53/99 with 750 nM hemagglutinin (HA) displayed a nice hemagglutination. The interference on the specificity of RBC and influenza virus was observed by anti-influenza aptamer at the concentration 31 nM, however, there is no hemagglutination prevention was noticed in the presence of complementary aptamer sequences. Spiking GNP conjugated Neu5Acα2,6Gal or Neu5Acα2,3Gal or a mixture of these two to the reaction promoted the hemagglutination to 63 folds higher with 12 nM virus, whereas under the same condition the heat inactivated viruses were lost the hemagglutination. Neuraminidases from Clostridium perfringens and Arthrobacter ureafaciens at 0.0025 neuraminidase units are able to abolish the hemagglutination. Other enzymes, Glycopeptidase F (Elizabethkingia meningoseptica) and Endoglycosidase H (Streptomyces plicatus) did not show the changes with agglutination. Obviously, sialyl-Gal-terminated glycan conjugated GNP amendment has enhanced the specificity of erythrocyte-influenza virus and able to be controlled by aptamer or neuraminidases. This article is protected by copyright. All rights reserved.
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) glycoprotein gene of Newcastle disease virus (NDV) variant strain V4(UPM) was determined by direct genomic RNA sequencing and confirmed by cycle sequencing. The gene comprises 1996 nucleotides encoding a 615 amino acid protein of size 67.4 kDa. The nucleotide and amino acid sequences of this strain were compared with those of the parent strain V4(QUE). There are 16 nucleotide substitutions on V4(UPM), eight of which are silent mutations and another eliminated a potential Asn-linked glycosylation site in V4(UPM). In addition, an Arg (403) residue was shown to be absent in the variant strain. This deletion is thought to be significant because of its location in a highly conserved region of the HN protein.
In comparison to the extensive characterization of haemagglutinin antibodies of avian influenza virus (AIV), the role of neuraminidase (NA) as an immunogen is less well understood. This study describes the construction and cellular responses of recombinant fowlpox viruses (rFWPV) strain FP9, co-expressing NA N1 gene of AIV A/Chicken/Malaysia/5858/2004, and chicken IL-12 gene. Our data shows that the N1 and IL-12 proteins were successfully expressed from the recombinants with 48 kD and 70 kD molecular weights, respectively. Upon inoculation into specific-pathogen-free (SPF) chickens at 105 p.f.u. ml-1, levels of CD3+/CD4+ and CD3+/CD8+ populations were higher in the wild-type fowlpox virus FP9 strain, compared to those of rFWPV-N1 and rFWPV-N1-IL-12 at weeks 2 and 5 time points. Furthermore, rFWPV-N1-IL-12 showed a suppressive effect on chicken body weight within 4 weeks after inoculation. We suggest that co-expression of N1 with or without IL-12 offers undesirable quality as a potential AIV vaccine candidate.
Avian Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are the most important pathogens for the poultry industry in recent times. The haemagglutinin protein and neuraminidase, serves as the target for the immune response of the host. Due to recurrent genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not withstand pressure by the novel hybrid virus. The emergence of genetic engineering technology provided the industry with new methods of manufacturing diagnostics tools and vaccines. After extraction of RNA from the cell culture of strain influenza A/Chicken/Malaysia/2004(H5N1) of AIV, the viral RNA was converted to cDNA by a specific primer. The cDNA was amplified by the polymerase chain reaction (PCR) and analyzed
by agarose gel electrophoresis. The intact PCR product of full length haemagglutinin gene was cloned in TO POTM TA Cloning vector. The full-length HA-encoding gene of H5N1 AIV was subcloned into a pPICZA vector. After successful ligation, the constructed plasmid was transformed into E.coli.Top10, Plasmid DNA from transformed bacteria was extracted in white colony and positive clones were confirmed by restriction digestion with Sacl and Not1 restriction enzymes, colony PCR screening and nucleotide sequencing. Construction of a recombinant pPICZA/H5HA plasmid containing the full length haemagglutinin gene was achieved as a first step
towards the expression in Pichia pastoris.