Analysis of the fusion protein gene of Newcastle disease viruses isolated in Japan.

The complete nucleotide sequences of the fusion (F) protein gene of Newcastle disease viruses (NDV) isolated in Japan from 1930 to 2007 (45 strains total) were determined and genetically analyzed. In the deduced amino acid sequences of fusion protein, the 5 potential asparagine-linked glycosylation sites and 10 cysteine residues were all conserved in the NDV examined in this study. The major epitopes involved in virus neutralization are conserved in most of the NDV strains isolated in Japan except a few strains. By virus neutralization test, no major antigenic differences were observed among representative strains of each genotype in Japan. All chickens vaccinated with the B1 strain survived without clinical signs after challenge with 2 NDV strains isolated in Japan (velogenic strains, JP/Ibaraki/2000 and JP/Kagoshima/91), which possess amino acids substitutions involved in virus neutralization in the F protein gene.

A novel genotype of avian infectious bronchitis virus isolated in Japan in 2009.

Avian infectious bronchitis viruses (IBVs) isolated from commercial layer flocks kept in Ibaraki Prefecture in 2009 were genetically and serologically characterized. Reverse transcription-PCR coupled with direct nucleotide sequencing and GenBank BLAST database analysis of the hypervariable region of the S1 subunit of the virus spike gene showed that these isolates are genetically very different from the previously known IBV genotypes in Japan. Furthermore, none of the antisera used in this study neutralized the index isolate (JP/Ibaraki/168-1/2009) in virus neutralization tests. These results suggest that the isolates are a novel IBV genotype in Japan (designated JP-IV).

Generation of monoclonal antibody that distinguishes PrPSc from PrPC and neutralizes prion infectivity.

To establish PrP(Sc)-specific mAbs, we immunized Prnp(-/-) mice with PrP(Sc) purified from prion-infected mice. Using this approach, we obtained mAb 6H10, which reacted with PrP(Sc) treated with proteinase K, but not with PrP(Sc) pretreated with more than 3 M GdnHCl. In contrast, reactivity of pan-PrP mAbs increased with increasing concentrations of GdnHCl used for pretreatment of PrP(Sc). In histoblot analysis, mAb 6H10 showed a positive reaction on a non-denatured histoblot but reactivity was lower when the histoblot was pretreated by autoclaving. Epitope analysis suggested that the extreme C-terminus of PrP is likely to be part of the epitope for mAb 6H10. MAb 6H10 immunoprecipitated PrP(Sc) from brains of mice, sheep, and cattle infected with prions. Furthermore, pretreatment of purified PrP(Sc) with mAb 6H10 reduced the infectious titer more than 1 log. Taken together, these results suggest that mAb 6H10 recognizes a conformational epitope on PrP(Sc) that is related to prion infectivity.

Characterization of prion susceptibility in Neuro2a mouse neuroblastoma cell subclones.

In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrP(Sc)) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrP(C)) at levels similar to the parental N2a cells. In contrast, there was a variation in PrP(C) expression in unsusceptible N2a subclones. For example, subclone N2a-1 expressed PrP(C) at the same level as the parental N2a cells and prion-susceptible subclones, whereas subclone N2a-24 expressed much lower levels of PrP mRNA and PrP(C) than the parental N2a cells. There was no difference in the binding of PrP(Sc) to prion-susceptible and unsusceptible N2a subclones regardless of their PrP(C) expression level, suggesting that the binding of PrP(Sc) to cells is not a major determinant for prion susceptibility. Stable expression of PrP(C) did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion-unsusceptible N2a subclones that expressed PrP(C) at levels similar to prion-susceptible subclones, indicated that a host factor(s) other than PrP(C) and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion-susceptible and -unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation.

Cell-surface retention of PrPC by anti-PrP antibody prevents protease-resistant PrP formation.

The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrP(Sc)), is essential for prion propagation. Antibodies to the C-terminal portion of PrP are known to inhibit PrP(Sc) accumulation in cells persistently infected with prions. Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrP(Sc) formation reduced PrP(Sc) accumulation in cells persistently infected with prions. The 50% effective dose was as low as approximately 1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrP(C)) expressed on the cell surface. Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb-PrP(C) complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggested that anti-PrP mAb antagonizes PrP(Sc) formation by interfering with the regular PrP(C) degradation pathway.