The Isolation And Identification Of PPV-SC1 And The Clone And Sequence Analysis Of Its Nonstructural Protein NS1

Porcine parvovirus (PPV) is a major cause of reproductive failure in swine, manifested as embryonic resorption, fetal mummification, abortion and stillbirths .The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. PPV infection has been increasing in China now. It is important for Chinese stockbreeding to identify ,prevent and control the PPV infection.In this research, we isolate one porcine parvovirus(ppv) strain successfully from the embryonic resorption ,fetal mummification .abortion and stillbirth which can reproduce on swine kidney primary cell,PK-15,ST and IBRS-2 cell lines ,and produce prominence CPE: the infected cells appear gathering and amalgamation.This isolate strain can agglutinate the RBC of guinea pig ,and can be inhibited by the positive ppv serum. Fluorescence assay is used to detect the antigens of porcine parvovirus(PPV),with bright green fluorescence in the infected cells .Under electron microscope ,ppv is 23nm in diameter.without envelope. According to these asseys, we isolated a porcine parvovirus (ppv) strain, named ppv-scl.One pair primers are designed according to the referried genome of ppv NADL-2 strain by Oligo6.0.After amplying a 2.2kb fragment form the PPV-SC1 RF-DNA,we clone the fragment into pMD 18-T,named pTNSl.The whole sequence which is 1989 bp long was determined by sequencing, including the complete ORF of PPV-SC1 NS1 which encoding 662 amino acids.Alignment of pairs of sequence indicates that there are 98% and 99% similary with other porcine parvovirus strains Kresse and NADL-2, respectively. Multiple sequence alignment discloses that there are a few difference between ppv-scl nsl gene and other ppv nsl gene: A-G at 39nt,T-C at 153nt,A-G at 175nt, A-C at 1117nt, A-C at 1535nt .Alternative codon in ppv-scl nsl have distinctly different frequentfy by codonbias analysis at EMBOSS(http://genopole.toulouse.inra.fr/bioinfo/emboss). Thereis not distinct hydrophobicity and transmenbrane helices in ppv-scl nsl protein. Struction domain anslysis of PPV-SC1 NS1 protein indicate that there are a ATP/GTP-binding site motif A (P-loop) at 398-405,16 Protein kinase C phosphorylation site,21 Casein kinase II phosphorylation site,and 3 cAMP/cGMP-dependent protein kinase phosphorylation site.At the same time ,there is a same motif between ppv-scl nsl and Poxvirus D5 protein-like which may share in the same fuction which is necessary during virion duplication.