Evaluation Of Newcastle Disease Virus Chimeras Expressing The Hemagglutinin-Neuraminidase Protein Of Velogenic Strains In The Context Of A Lentogenic Recombinant RLaSota Virus Backbone

Newcastle disease (ND) is one of the most serious in fectious diseases of poultry, and virulent ND outbreaks . NDV, the causative agent of ND,is anon-segmented, single-stranded, negative sense RNA virus that belongs to the genus Avulavirus within the Paramyxovirinae subfamily of the Paramyxoviridae family. Its genome is approximately 15kb in length and encodes six proteins, namely, nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), Hemagglutinin- Neuraminidase (HN) and large protein (L). Most of investigation has been focused on the contributions of the F and HN proteins to pathogenicity,and a major factor in the pathogenicity of Newcastle disease virus (NDV) is the amino acid sequence of the fusion protein cleavage site.The HN protein of NDV is a multifunctional protein.The HN protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. It possesses both the receptor recognition and neuraminidase (NA) activities associated with the virus. It recognizes sialic acid-containing receptors on cell surfaces; it promotes the fusion activity of the F protein, Thus, the HN protein plays an important role in viral infection.In this study, we exchanged the HN genes of the velogenic strains,F48E9,HB38 by use of reverse genetics procedures, the HN genes of the velogenic strains , F48E9 and HB38, and an avirulent recombinant NDV strain, rLaSota,were exchanged. Briefly,viral genomic RNA, F48E9 and HB38, was obtained by using the Trizol reagent, following manufacturer'srecommendations(Invitrogen).The complete antigenome of the NDV strain,F48E9 and HB38,were generated by reverse transcriptase polymerase chain reaction (RT-PCR), and cloned into a modified pBluescript-based vector by taking advantage of unique restriction enzyme sites , MunIand SpeI. The plasmid was modified to contain the sequence of HN gene,F48E9 and HB38.Viral rescue from infectious clone, rL-F48E9HN and rL-HB38HN. Sequence analysis of rescued viruses, Viral RNAs were extracted fromallantoic fluid of the rescued virus infected embryonated chickeneggs.Growth characteristics of viruses in embryonated specific-pathogen-free(SPF) chicken eggs.Growth characteristics of the parental and chimeric viruses was concordant,opposite to F48E9 and HB38.Biological activities of mutant viruses.We analyzed whether the origin of the HN protein determines the biological activities of NDV. The chimeric iruses showed 51%,62% increase in HAd activity.The fusogenic abilities of the parental and chimeric viruses did not differ significantly.Pathogenicity studies of mutant viruses. It wanted to determine whether the differences in in vitro biological characteristics of the chimeric viruses would translate into increased or decreased virulence in chickens or chicken embryos.The chimeric viruses , rL-F48E9HN and rL-HB38HN,with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), Intravenous pathogenicity index ,indicating that virulence is a function of the amino acid diff-erences in the HN protein. rL- F48E9HN and rL- HB38HN MDT(148h,120h);ICPI(0.43,0.64) ;IVPI(0).Results of the research performed to elucidate the contribution of the HN protein, chimeras rL-F48E9HN and rL-HB38HN,showed a significant increase in pathogenicity, which failed to increase the virulence of these viruses from their- pre-recombination pathotype to a higher one.These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavabili-ty of F protein alone does not determine the virulence of a strain.