Newcastle Disease Virus Neuraminidase Inhibitions: Differences Among Strains and a Proposed Mechanism for the Elution- Inhibition Antibody Reaction

Newcastle disease virus (NDV) neuraminidase (NA) inhibition with elution-inhibition (EI) antibodies was estimated with the EI reaction using strain 575 isolated from a mute swan (Cygnus olor). The NA inhibition was assessed by persistent red blood cell (RBC) agglutination patterns, rather than by the standard test involving the reduction in the cleavage of added artificial substrates. The more numerous antibody determinants for the hemagglutinin (HA) of strain 575 enriched the antiserum for EI antibodies. Thus, the proposed mechanism for NA inhibition is governed by the relative amounts of HA and NA antibody determinants. The NA of strains Cg and Roakin, but not the B1 vaccine, were inhibited only if the antiserum was enriched for EI antibody with strain 575. The antiserum contained complement-dependent antibody. The EI antibody determinants were Intern J Appl Res Vet Med • Vol. 3, No. 3, 2005 Intern J Appl Res Vet Med • Vol. 3, No. 3, 2005 234 (NDV) anti-neuraminidase (NA) elutioninhibition (EI) antibodies was used to estimate prevalences. The populations tested were European mute swan (Cygnus olor), tundra swan (Cygnus columbianus), and Canada geese (Branta canadensis). Differences in EI antibody prevalence were found between adult mute and tundra swan as well as between tundra swan and Canada geese. Seroconversions as well as persisting or declining titers were found in mute swan. Thus, using the field strain 575 isolated from a mute swan, the differences in individuals and populations were delineated. The EI assay that employs persistent red blood cell (RBC) agglutination patterns is less complicated than the standard test, is inexpensive, and is suitable for testing large numbers of polyclonal sera. The purpose of this report is to examine how strain 575 functions. Two explanations for the NA inhibition will be explored. One possibility is that the swan strain has more hemagglutinin (HA) determinants than strains that failed to respond to EI antibody. Enrichment for EI antibodies would result in NA inhibition. Another possibility is that the infrequent HA-sialic acid configuration required for the EI reaction occurs only with the strain 575. Enriching antiserum for EI antibody with the swan strain and then testing if the NA of other strains could be inhibited was the approach for resolving the possibilities. The reactions leading to antibody enrichment and NA inhibition are described. Additionally, combining NA activities with the results from the indirect antibody determinant titrations provide numerical values that help characterize the NA variation among the 4 strains examined. MATERIALS AND METHODS NDV Strains, Polyclonal Antiserum, RBC, and Clinical Signs Partial purification of PMC-1/Mute swan/Maryland/575/1977 (strain 575) with sephadex filtration and centrifugation has been described. Biologic plaque purification on chicken embryo cells and terminal endpoint dilution tests in embryonating eggs showed that strain 575 is genetically stable. The Roakin strain and homologous, post-infection chicken antiserum were obtained from the Centers for Disease Control (CDC), Atlanta, GA. Ten NDV strains were recovered from 4 avian species. The 17th passage in embryonating eggs of the B1 vaccine and the 13th passage of the virulent Cg 179 (Cg) strains were obtained from the late F. B. Bang, The Johns Hopkins University, Baltimore, MD; he also donated sera from rabbits immunized with the Beaudette B strain. The Beaudette B strain, like Cg, is a strain lethal for chicken embryos. Rabbit sera were drawn 17 days post-immunization and contained IgG and IgM antibodies; either will mediate the HAinhibition (HI) and EI reactions. Red blood cells from 2 leghorn chickens were selected for the tests by trial and error and suspended (0.3% v/v) in phosphate (0.05 M) buffered (pH 7.2-7.4) NaCl containing 0.1% NaN3 (PBS). Before testing, all antisera were adsorbed with RBC. All birds were without signs of infection. The flightless, juvenile 575 swan was resighted with no apparent clinical disease during 24 months following isolation of the strain from feces. No deaths occurred in 72 hours among embryonated eggs inoculated with strain 575. Serologic Tests, Localization of NDV on RBC With Normal RBC and Fluorescent Antibodies, and Tests for NDV in Antiserum Inhibition of NA was estimated in a direct assay by persistent agglutination patterns of RBC rather than indirectly by a reduction in the cleavage of added artificial substrates. Strain 575 elutes from RBC above the level of effective EI antibody. Following the incomplete elution, the RBC became sensitized. The NA on sensitized RBC is inactive whereas the HA is functional and agglutinates up to 7 volumes of normal RBC in the agglutination-separation (AS) reaction. During 37 ̊C incubation