Investigation Of The Epidemiology Of Porcine Transfusion Transmitted Virus And Expression Of Cap Protein And Contruction Of Infectious Clone

Porcine transfusion transmitted virus(PTTV)or Porcine torque teno virus(PTTV)has two kind of genotypes: PTTV1 and PTTV2.The role of both PTTV1 and PTTV2 in the pathogenesis of specific swine disease remains debatable.However, recent reports suggested the coinfection of PTTV with porcine circovirus type 2(PCV2)that may causes postweaning multisystemic wasting syndrome(PMWS).To investigate the co-infection situation of porcine PTTVs and PCVs, 280 samples from 14 provinces of China were detected by PCR. The results indicated that the positive rate for PTTV1 and PTTV2 infection were 51.8% and 28.2%, respectively, and 18.2% for PTTV1 and PTTV2 co-infection. The positive rate of PCV1 and PCV2 infection was 41.1% and 37.5%, respectively, and 19.6% for PCV1 and PCV2 co-infection. In addition, the co-infection rate of PTTV1 and PTTV2 reached up to 75.0% in PCV2 positive samples. Moreover, two PTTV1 strains genomes were cloned and sequenced, and the similarity of nucleotide acid sequences was 67.3% to 95.1% compared with six PTTV1 genomic sequences in GenBank. The similarity of 4 PTTV2 strain genomic sequences was 84.7% to 90.4% compared with four PTTV2 genomic sequences in GenBank. However, the similarity of genomic nucleotide acid between PTTV1 and PTTV2 was only 44.0%. The high variation regions of PTTV1 and PTTV2 were located at 520 nt to 2594 nt and 720nt to 2170 nt, respectively. These data indicated that the co-infection of PTTV and PCV exists in pig herds of China, and high variation and genetic diversity are present in the different PTTV1 and PTTV2 strains. They maybe contributed to clinical diseases cooperatively.Both PTTV1 and PTTV2 are similar to each other in genome containing three major open reading frames(ORFs), which are ORF1,ORF2 and ORF3. The ORF1 is considered to encoding the capsid protein(Cap)of the virus. The ORF2 gene may be encoding a protein which is essential for the replication of TTV DNA, while ORF3 maybe encoding a protein that is assoicated with apoptosis of cells.As the Cap is the major structure protein of vrius, it can stimulate the body to induce humoral immune response and cell immune response, so it is an ideal target antigen. In order to express the capsid protein of PTTV2, PTTV2-ORF1 gene was amplified by PCR and was cloned into prokaryotic expression vector pGST-6p-1.The recombinant plasmid was transformed into E.coli. Rosetta (DE3) cells, and then the fusion protein (GST-PTTV2-Cap) was expressed in the competent cells after induced with a final concentration of 1 mmol /ml of IPTG for 3 h.The expressed protein was approximate 82 kDa and existed in inclusion body form. Western-blot analysis showed that both GST tag and fusion protein could react with anti-GST monoclonal antibody.The PTTV Cap recombinant protein expression is the first report, it provides a foundation for establishing serological diagonistic method for the virus.The reason is that the prokaryotic expression system can not processing or folding the protein correctly, while the eukaryotic expression system can overcome that short. In this study, the full-length and the partial gene of PTTV2-ORF1 were amplified by PCR and cloned into pBlueBac4.5/V5-His TOPO TA vector. The recombinant plasmid (pBlueBac4.5/V5-His-PTTV2-ORF1-F and pBlueBac4.5/V5-His-PTTV2-ORF1-S) and Bac-N-BlueTM DNA were co-transformed into the insect cell (Sf21). Two kinds of recombinant baculaviruses of rBlueBac-PTTV2-ORF1-F and rBlueBac-PTTV2-ORF1-S were harvested after three circles of plaque cloning. The two kinds of recombinant fusion capsid proteins with a molecular weight of 79 kDa and 43 kDa were analysied using SDS-PAGE and Western blot. As there has a six histidines tag at the C terminal of the recombinant fusion capsid protein, its makes easier to purify the protein by Ni2+ affinity chromatography.The purified Cap protein could be used as antigen to further establish seriogocal diagnostic method for the virus.Up to now, PTTV has not been cultured in any cells. In order to obtaining the vrius, the full-length genome of PTTV2 was amplified by PCR, after the genome was digested by restriction enzyme and self-circularization. The PTTV2 genome was transfected into ST and PK15 cells, respectively. PTTV2 DNA was dectected by PCR for the rescued virus after passages in the cells. The PCR results were positive within from 1th to 6th passages, and then changed into negative reaction after the 7^thto 9^th passages. The study suggests that ST or PK15 cells may not be suitable for PTTV replication or proliferation.