The Role Of HN Protein In Virulence And Tissue Tropism Of Newcastle Disease Virus

Newcastle disease (ND) is a worldwide devastating disease of poultry caused by Newcastle disease virus (NDV). After four pandemics in the world, its host ranges have been significantly enlarged. Aquatic birds are usually resistant even to the most virulent NDV strains against chickens. However, outbreaks caused by NDV in waterfowl have been reported in various regions of South and East China since the late 1990s. In recent years, the spread of viruses in goose flocks in China has raised concerns about their emergence, characteristics and evolution. The Hemagglutinin-neuraninidase (HN) protein of NDV is a multifunctional glycoprotein which mediates the attachment of virus particles to sialic acid-containing receptors of the host cell, promotes the fusion activity of the F protein and also acts as neuraninidase by removing the sialic acid from progeny virus particles. Although the functions of the HN protein in NDV infection have been well studied, its role in NDV pathogenesis still remains uncertain at present.In this study, the velogenic NDV vector backbone was firstly used to produce two chimeras coding for the HN protein of the more virulent genotype VII strain I4 and lentogenic genotype II strain Clone30, respectively. Subsequently, the biological characteristics of the recombinant viruses were further studied for elucidating the role of HN protein in virulence and tissue tropism.1 The construction and rescue of HN chimeric virusesUsing PCR mutagenesis, the restriction site AflII (nt 2456) of R123 was eliminated. As a result, the unique restriction sites AflII (nt 3279)and SspI (nt 5672) were employed to substitute the HN gene with that from the strain I4 and Clone30. The resulting plasmids were designated pZJ1/I4HN and pZJ1/ClonHN. The fragment containing the green fluorescence protein gene (GFP) was then inserted into the plasmids pZJ1/I4HN and pZJ1/ClonHN between P and M gene, respectively to generate two GFP-tagged constructs of pZJ1/I4HN-GFP and pZJ1/ClonHN-GFP.These four chimeric plasmid constructs were eventually used to recover the recombinant viruses: rZJ1/I4HN, rZJ1/ClonHN, rZJ1/I4HN-GFP, rZJ1/ClonHN-GFP by transfection. Sequence analysis of the chimeric viruses confirmed the presence of substituted HN genes and the insertion of GFP gene.2 The effect of HN protein on the biological properties of the chimeric viruses.2.1 The effect of HN protein on biological properties in CEF cells.Analysis of the multi-step growth curves, cytopathogenicity effects, plaque sizes and morphologies showed that these chimeric viruses reached similar titers, with equal kinetics, when compared to the parental virus. The kinetics of replication, plaque sizes and morphologies of rZJ1, rZJ1/I4HN and rZJ1/ClonHN were also similar, though the replication and yield of rZJ1/ClonHN were delayed slightly.2.2 The effect of HN protein on virulence of chimeric virusesThe virulence of the recovered viruses was determined by the MDT, ICPI and IVPI tests. In comparison with parental virus rZJ1, rZJ1/I4HN showed shorter MDT whereas rZJ1/ClonHN exhibited longer one, though their ICPI and IVPI were similar. The infection tests, performed in susceptible SPF birds which were inoculated with viruses by natural route, showed that the chimeric virus encoding the I4 HN gene exhibited higher pathogenicity in birds when compared to rZJ1, whereas rZJ1/ClonHN with HN gene from Clone30 had reduced virulence.2.3 The effect of HN protein on tissue tropism of chimeric virusesTo further determine the role of HN protein in NDV pathogenesis, tissue tropism of the chimeric viruses carrying GFP gene in SPF embryos and chicks were assessed. The results indicated that rZJ1-GFP and rZJ1/I4HN-GFP were distributed systemically in chicken embryos and chicks, while rZJ1/ClonHN-GFP remained in a few tissues with less extent of GFP expression. The results of the immunohistochemistry (IHC) showed that there were some differences in tissue distribution among these chimeric viruses. Except lung, spleen and brain, the parental virus rZJ1 was intensively distributed in liver, tracheae and kidney, while extensive and intensive viral antigen of the rZJ1/I4HN could be examined in most of tissues such as brain, liver, spleen, tracheae and kidney. On contrast, for chimeric virus rZJ1/ClonHN, the distribution of viral antigen was restricted in a few tissues with significantly less intensity. The results mentioned above suggested that the NDV HN protein play an important role in tissue tropism.Conclusion:The principal findings in this study are: (1) Heterologous pairs of HN and F proteins are fully functional; (2) In addition to the F protein cleavage site, the HN protein also contributes to the NDV virulence; (3) The origin of HN protein plays an important role in tissue tropism, and may influence efficient viral entry into host cells.