| Newcastle disease virus (NDV) is a member of the family Paramyxoviridae, and has been assigned to the genus Avulavirus in the subfamily Paramyxovirinae. It causes a serious respiratory and neurological disease in all species of birds and is an economically important infectious agent, causing substantial losses to the poultry industry.Cleavage of the F protein is known to be required for initiation of infection and is considered to be a major determinant of NDV virulence. The HN protein is also required for fusion to occur by cooperating with the F protein. Cleavage of the F protein of virulent NDV strains occurs by ubiquitous subtilisin-like proteases; whereas, cleavage in avirulent strains occurs by trypsin-like enzymes found in limited tissues. The cleavage efficience is different among most lentogenic, mesogenic and velogenic NDV strains. The F protein of all lentogenic strains has a monobasic cleavage site (112 (G/E) (K/R) Q (G/E) RL117), which is cleaved by extracellular proteases restricted to specific tissues; whereas, the F protein of all mesogenic and velogenic NDV strains has a multibasic cleavage site (112 (R/K) RQ (R/K) RF117), which is cleaved by ubiquitous intracellular proteases. Since the F protein cleavage efficiency has long been considered to be a major determinant of NDV virulence, The HN protein of NDV is a multifunctional protein. It possesses both the receptor recognition and neuraminidase (NA) activities associated with the virus. Although the functions of the HN protein in NDV infection have been well studied, its role in NDV pathogenesis is not known at present. The advent of reverse genetics technology has revolutionized the field of RNA viruses. It is now possible to manipulate even negative-stranded RNA viruses at will, and evaluate the effects of these changes on the biology and pathogenesis of these viruses. In this study, we have used our full-length NDV cDNA clone to generate infectious NDV with defined mutations in the F0 protease By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR↓L) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F) and (GRRQRR↓L). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] 0.36). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI 1.28 out of a possible maximum of 2.0). Cells infected with the different strains followed by indirect immunofluorescence, results showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. This study with recombinant NDV generated by reverse-genetics techniques showed that modification of a lentogenic F cleavage site to a velogenic cleavage site increased the virulence of the strain but did not reach the virulence level of velogenic strains. This result indicated that the efficiency of cleavage of the F0 protein is not the sole determinant responsible for the virulence of NDV.We also exchanged the F gene of rLaSota with the F gene from Mukteswar,F48E9 and HB38,Recovered virus from these full length of cDNA was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth characteristics in embryonated eggs. Moreover, an ICPI of 1.17 (rL-MuF), 0.83(rL-F48F), 1.18(rL-HBF) was obtained for virus as determined by intracerbral inoculation of day-old SPF chickens, proving that the F protein of NDV is not yet the only determinant fator for the virulence of NDV.The surfaces of NDV particles contain two important functional glycoproteins: the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of an avirulent recombinant NDV strain, rLaSota, were submited by that of Mukteswar, a virulent NDV strain. The hemadsorption of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The chimeric viruses with reciprocal HN proteins lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos, indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.Recently experiments using foreign proteins demonstrated that NDV will accommodate foreign genetic material and stably maintain it during serial passages in cell culture and embryonated eggs, but the recombinant NDV generated from full-length cDNA were growth retardation and attenuation. To study the effection of foreign proteins for the virulence of NDV, We insert different foreign gene between the M gene and F gene based on the mutational backbone of rL-F (M) F. The reporter gene encoding enhanced green influence protein (EGFP),the VP2 gene of infectious bursal disease virus (IBDV) and HA gene of H5N1 highly pathogenic avian influenza virus (HPAIV) were inserted as an additional transcription unit between P and M gene in the NDV genome. Replication kinetics of the rNDV viruses in SPF embryonated eggs showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was glycoprotein. The MDT and ICPI also showed a decrease compared with the parent strains. These results show that foreign genes inserted in the NDV genome will affect replication efficency or virus virulence. |
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