Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2(13) HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.
Avulavirus* ; Baculoviridae* ; Blotting, Western ; Clone Cells ; Cross Reactions ; Diagnosis ; Ducks ; Glycosylation ; Hemagglutination ; HN Protein ; Immune Sera ; Insects ; N-Acetylneuraminic Acid ; Serologic Tests ; Serotyping ; Spodoptera

Oroxylum indicum has long been used in Asian traditional medicine to prevent and treat respiratory diseases, diabetes, diarrhea and other conditions. Oroxylin A is a flavone that is present in Oroxylum indicum and in Scutellaria baicalensis. Because the root extracts of both plants have been shown to have anti-allergic effects, the authors investigated whether oroxylin A is likely to have beneficial effects on allergic asthma using female Balb/c mice and rat RBL-2H3 mast cells. Antigen-induced degranulation was measured in vitro by measuring β-hexosaminidase activity. A murine ovalbumin-induced allergic asthma model was used to test the in vivo efficacy of oroxylin A. Sensitization and challenge of ovalbumin induced allergic asthma responses, the accumulations of eosinophils and Th2 cytokine levels in bronchoalveolar lavage fluid and lung tissues. Oroxylin A administration decreased numbers of inflammatory cells, especially eosinophils, and reduced the expression and secretion of Th2 cytokines, including IL-4 and IL-13, in lung tissues and bronchoalveolar lavage fluid. Histologic studies showed oroxylin A reduced inflammatory signs and mucin production in lungs. These findings provide evidence that oroxylin A has potential use as an anti-allergic therapeutic.
Animals ; Asian Continental Ancestry Group ; Asthma ; Bronchoalveolar Lavage Fluid ; Cytokines ; Diarrhea ; Eosinophils ; Female ; Humans ; In Vitro Techniques* ; Interleukin-13 ; Interleukin-4 ; Lung ; Mast Cells ; Medicine, Traditional ; Mice ; Mucins ; Ovalbumin ; Rats ; Scutellaria baicalensis

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.
Animals ; Antibodies, Viral/*blood ; Antigens, Viral/*diagnostic use/genetics/metabolism ; Baculoviridae/genetics ; Chickens ; HN Protein/*diagnostic use/genetics/metabolism ; Hemagglutination Inhibition Tests/*methods/veterinary ; Newcastle Disease/*diagnosis/immunology/virology ; Newcastle disease virus/genetics/*immunology/metabolism ; Poultry Diseases/*diagnosis/immunology/virology ; Recombinant Proteins/diagnostic use/genetics/metabolism ; Sf9 Cells ; Spodoptera

A neutralization-resistant mutant of Newcastle disease virus (NDV) Kr005 strain belonging to class II genotype VII was generated using a neutralizing monoclonal antibody and its biological effects were assessed. The mutant showed single amino acid substitution (E to K) at position 347 of the hemagglutinin-neuraminidase (HN) protein (E347K mutant). The E347K mutant exhibited marked rounding of the cells and few syncytia in infected chicken embryofibroblast (CEF) cells. The hemadsorption and neuraminidase activities of the E347K mutant of the wild-type virus were 118% and 166%, respectively. The mutant produced a rapid elution pattern whereas the wild type had a slow elution pattern. Growth kinetics studies showed that the E347K mutant produced an 80-times higher yield of extracellular virus in CEF cells compared with the wild-type virus. The time-course virus titer showed a marked increase in mutant-infected cells from 6 h to 12 h post infection (pi), which was consistent with the titer pattern time-course for NA activity. The E347K mutant virus showed a slight decrease in virulence compared to the wild-type virus, but there was no change in pathotype when measured by in vivo pathogenicity testing. These results suggest that an E347K mutation in HN protein might be associated with increased NA activity and subsequent enhancement of virus release from infected cells without change in viral pathotype.
Amino Acid Substitution ; Animals ; Chickens ; Genotype ; Giant Cells ; Hemadsorption ; HN Protein ; Kinetics ; Neuraminidase ; Newcastle Disease ; Newcastle disease virus ; Sprains and Strains ; Viral Load ; Virus Release ; Viruses

To explore the anti-allergic and anti-inflammatory effects of extracts of Petasites genus, we studied the effects of s-petasin, a major sesquiterpene from Petasites formosanus (a butterbur species) on asthma and peritonitis models. In an ovalbumin-induced mouse asthma model, s-petasin significantly inhibited the accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar fluids. S-petasin inhibited the antigen-induced degranulation of beta-hexosamidase but did not inhibit intracellular Ca2+ increase in RBL-2H3 mast cells. S-petasin inhibited the LPS induction of iNOS at the RNA and protein levels in mouse peritoneal macrophages. Furthermore, s-petasin inhibited the production of NO (the product of iNOS) in a concentration-dependent manner in the macrophages. Furthermore, in an LPS-induced mouse model of peritonitis, s-petasin significantly inhibited the accumulation of polymorpho nuclear and mononuclear leukocytes in peritoneal cavity. This study shows that s-petasin in Petasites genus has therapeutic effects on allergic and inflammatory diseases, such as, asthma and peritonitis through degranulation inhibition in mast cells, suppression of iNOS induction and production of NO in macrophages, and suppression of inflammatory cell accumulation.
Animals ; Asthma* ; Eosinophils ; Leukocytes, Mononuclear ; Lymphocytes ; Macrophages ; Macrophages, Peritoneal ; Mast Cells ; Mice ; Peritoneal Cavity ; Peritonitis* ; Petasites ; RNA