Isolationand Identification Of Porcine Parvovirus And Parts Of Its Biological Characteristics

Porcine parvovirus (PPV) is one of the most important pathogens responsible for reproductive disorders of sows. This disease is world widespread and endemic in most pig farms,seriously affecting the development of the swine industry.In present study four virus strains were successfully isolated from abortion fetal produced by sows with the signs of the embryonic resorption,fetal mummification and abortion in some reproductive fallue pig farms. These strains could multiply in PK-15 cell lines and produce prominent cytopathic effect (CPE),agglutinate the 0.6% red blood cel(lRBC)of guinea pig and could be inhibited by positive PPV antiserum. Indirect immunofluorescence assay was used for detecting the antigens of PPV with bright greed fluorescence in the infected cells. Partial sequence of ns 1 gene was amplified by PCR and compared with that of other isolates in GenBank,which showed they had more than 98% nucleotide homology.They were identified as Porcine parvovirus and named as strains HNZK-1,HNZK-2,HNAY,and HNLH. Three pairs of primers which cover the whole genome of PPV were designed to amplify the whole length genome of strains HNZK-1 and HNAY by PCR. The sequencing results showed that the lengths of strains HNZK-1 and HNAY are 4668nt and 4610nt,respectively.There is no insertion or deletion in the coding region,. However, some deletions exist in the non-coding region nearby the vp 2 stop codon, which,compare with NADL-2,lack 15(strains HNZK-1) and 69(strains HNAY) nucleotides respectively. The sequences alignments of ns l and vp2 genes and their deduced anminal sequence showed that strains HNZK-1 and HNAY had high homology with strain Nanjing200802. The genetic variation analysis showed that the PPV strains prevailing in china adjoined each other. Strain HNZK-1 could resist ether,trypsin,pH3.0 acid,and 56℃heat treatment . When inoculated with PPV at the same time of the passage, cells produced a high HA titer. PPV of strain HNZK-1 was inactivated by formaldehyde to prepare oil emulsion vaccine. The vaccine could induce high HI antibody titer over 2¹⁰ in guinea pig. There was almost no decline in antibody titers 12 weeks after immunization. The antiboby titer of control vaccine was 2¹～2² lower than PPV HNZK-1 strain oilemulsion inactivated vaccine。Two primers specific for the structural protein gene of PPV were designed and a real-time polymerase chain reaction(real-time PCR) with SYBR GreenⅠwas developed for quantification detection of PPV. The detection limit of the samples by SYBR GreenⅠreaction was 12 PPV copies·reaction^-1.A positive linear relationship could be seen in a 10⁸ dilution range of template concentrations. Other porcine pathogens involved in reproductive disorders including porcine circovirus type 2 (PCV 2), porcine reproductive and respiratory virus (PRRSV), Japanese encephalitis virus type b (JEV), classical swine fever virus (CSFV) and Aujeszky's disease virus(PRV) were not detected by this assay. Dynamic Analysis for porcine parvovirus replication in vitro(25cm² cell culture flask)by this assay traced a curve of PPV proliferation and the results indicated that the amount of the extracellular virus copies gradually decreased from 0 h to 36 h post inoculation, and then began to increase. The copies of virus in the cultural supernatants (1.739×10¹⁰ copies) were higher than that in the cells (1.321×10¹⁰ copies) from 84 h on post inoculation. The copies produced a peak 108 h post inoculation (7.626×10¹⁰ copies) and then rapidly reduced. While intracellular virus particles showed a logarithmic growth phase within the first 24 h post inoculation followed by a slow growth until 72h post inoculation(1.425×10¹⁰ copies), and reached a peak of titer and maintained a high level until 108 h post inoculation. CPE corresponding to the process of the virus growth was characterized by the cell aggregation and about 80% plaque formation. With the large number of cell death, copies of both intracellular and extracellular virus began to sharply reduce at 108 h after inoculation.The main antigen domain for vp 2 gene of PPV strain NHAY was amplified by a PCR method using a pair of specific primers designed according to the published nucleotide sequences in GenBank.The PCR products were cloned into pMD-18T Easy vector and an 873bp nucleotide fragment was acquired. Then the vp 2 gene of strain HNAY was cloned into the vector pET-32a(+), and the recombined prokaryotic expression plasmid,pET-vp2,was successfully constructed and transformed into E.coli BL21(DE3).Inducted by IPTG,the fusion protein was highly expressed in E.coli BL21(DE3).The recombinant protein with a molecular weight about 49 KDa was approved with immunological activity by SDS-PAGE and Western- blotting.