Molecular Biological Studies On RRSV & NDV-analysis-software Development

1. The first part of present study was focused on the functions of structural protein P9and nonstructural proteins Pns6 and Pns10 of rice ragged stunt virus (RRSV). RRSV is a typical member of the genus oryzavirus within the family Reoriridae, causingrice diseases in Southeast Asia and South China. It is disseminated by the insect vector, therice brown planthopper (Nilaparvata lugens) and can replicate in both its plant host, rice, andinsect vector. RRSV has a complex multi-component particle bearing spike on its outersurface and its genome contains 10 segments dsRNA. (1) Transgenic rice lines expressing the 39 kDa spike protein showed good resistance toinfection by RRSV. Furthermore, N. lugens fed on these plants prior to feeding onRRSV-infected plants were significantly protected against RRSV infection. The viral titre ininsects initially fed on transgenic plants and then on RRSV-infected plants was inverselyproportional to the levels of the 39 kDa protein expressed in the transgenic plants. Thissuggests that the 39 kDa protein interferes with the interaction between the intact virusparticles and insect cell receptors and that the spike protein of RRSV contributes to vectorspecificity. This approach would probably be a more environment-friendly and sustainablemethod of virus control than by actual eradication of insect vectors. (2) Pns6, a nonstructural protein of RRSV encoded by segment 6, has sequencenonspecific nucleic acid binding activity, showing a preference for single-stranded nucleicacid. The stability assay showed the Pns6:ssRNA complex is stable within the 250 mM NaCland only 50% of the complex was dissociated even in the 600 mM NaCl. The heating of Pns6at 60 °C and 80 °C for 5 min did not affect its RNA-binding activity indicating thus theactivity was quite stable at the temperature tested. By using five truncated derivatives of Pns6,it was found that the basic region from amino acid 201 to 273 of Pns6 was the unique nucleicAbstractacid binding domain. Subcellular fractionation of leaf tissues of RRSV-infected rice plantsand subsequent Western blotting had shown that Pns6 accumulated predominately in thecytoplasmic membrane fraction. Therefore it probably is not the movement protein of RRSV. (3) RRSV nonstructural protein 10 (Pns 10) encoded by viral genomic segment S10contains amino acid sequence motifs typical of ATPase proteins. In order to examine theATPase activity of the RRSV Pns10, the second major ORF of S10, encoding Pns10( spanning nucleotides 142 to 1032) was fused with chitin binding domain (CBD). The fusionprotein was expressed in E.coli and purified with chitin beads. The purified Pns10 withoutCBD fusion part possessed ATPase activity. Characterizations of the ATPase activity of Pns10showed that the activity had an optimum pH of 8.0, an optimum temperature of 47 and anoptimum divalent-cation concentration of 1 mM MgCl2 or CaCl2. All NTPs and dNTPs couldbe hydrolysed. The point mutation (K20Q) at consevative motif A could abolish the ATPaseactivity of Pns10. This study reveals for the first time an NTPase activity associated with aphytoreovirus-encoded protein. 2. The second part of this study was focused on the development of a novel fasttechnique to differentiation of vaccine and wild strains of Newcastle disease virus,simultaneously determinating their virulence and genotype. Newcastle disease (ND), caused by Newcastle disease virus (NDV), is one of the mostserious diseases of poultry. NDV is the unique member of avian paramyxovirus serotype-1(APMV-1). Its genome is a minus strand of ssRNA which includes about 15000 nucleic acidsencoding six genes, 3'-NP-P-M-F-HN-L-5'. The F Protein is the key factor for the fusionbetween the virus and host cell. The virus virulence is mostly determined by the amino acidsequence of cleavage site of the F protein. According to the molecular phylogenetic of the Fgene, all NDV strains could be separated into seven genotypes.