Molecular Epidemiology Of Class â…  Newcastle Disease Virus And Establishment Of Reverse Genetics System Of NDV08-004 Strain

The full length NP and P gene of 60 ClassⅠNDV isolates, obtained in China during 2008 to 2010, were amplified and sequenced. The full-length infectious cDNA clone of NDV08-004 strain was also constructed and the recomninant virus rNDV08-004 was rescued successfully.This provide a foundmation for conducting study on pathogenesis and vaccines of classⅠNDVs.1 Molecular Characterization of NP gene of 60 ClassⅠNDV isolates obtained from China during 2008 to 2010The NP gene of 60 classⅠNDV isolates, obtained from China during 2008 to 2010, were amplified by RT-PCR and sequenced. The complete NP gene of all 60 isolates was composed by 1746 nucleotides, which can code 489 amino acids. We found two special sites for NP gene (D460E and S468T) different between ClassⅠand classⅡNDV. Homology analysis revealed that the homology of amino acids of NP gene between classⅠand classⅡNDV strains were between 88.0%～92.7%. the homology of nucleotides of NP gene between classⅠand classⅡNDV strains were between 75.2%～79.5%. Phylogenetic analysis based on the complete NP gene showed that all isolates can be divided into two distinct genotypes, and the difference among the two genotypes was between 2.7%～4.1%.2 Molecular Characterization of P gene of 60 ClassⅠNDV isolatesThe P gene of 60 classⅠNDV isolates were amplified by RT-PCR and sequenced. The complete P gene of all classⅠNDV isolates was composed by 1463 nucleotides, which can code 399 amino acids. Compared with classⅡNDV, the P gene of all classⅠNDV isolates were inserted with 12 nucleotides in the coding region. We found some special sites for P gene (E64D, G67D, N75Q, N81Q, E106D, F187Y, I245V, K304R, A311S, D340E, I354V and S374A) different between ClassⅠand classⅡNDV. Homology analysis of P gene revealed that the homology of amino acids between classⅠNDV isolate and classⅡstrains were between 64.5%～72.8%. The homology of nucleotides between classⅠNDV isolate and classⅡstrains were between 66.3%～72.2%. Phylogenetic analysis based on the complete P gene showed that all isolates can be divided into two distinct genotypes, which indicated the P gene may have a similar evolution characterization compared to NP genes. The difference of amino acid among the two genotypes was between 2.7%～4.1%.3 Construction of rescue system of classⅠNDV strain NDV08-0043.1 Establishment of full-length genome of NDV08-004 strain and helper plasmid of L geneSeven fragments of the genome were amplified and cloned into pGEM-T Easy Vector with the designed primers, and then subcloned into the LT-pOLTV5 using the single restriction sites consequently. The plasmid NDV08-004-po, which contained the full-length cDNA of NDV08-004 strain, was constructed successfully. In order to differenciate the recombinant strain from the wild-type virus, a molecular tag which have a muatation at the position of 7022nt of the genome (C→T) was involved. The similar strategy was also conducted to construct the helper plasmid pCI-L, which was subcloned the entire L gene into pCI-neo vector.3.2 The rescue of recombinant virus r-NDV08-004The plasmid NDV08-004-po together with three helper plasmids, pCI-NP, pCI-P and pCI-L, were cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculating the supernatant of the transfected cell culture into SPF embryo, recombinant virus NDV08-004 was successfully rescued. But the HA titer of the first generation rescued virus is only 21og2 the HA titer can get 71og2 by propagating. The success of the rescue virus was further proofed by sequencing the molecular tag. The work mentioned above provided a foundation for conducting research on pathogenesis and vaccine of classⅠNDVs.