Identification Of Transcripts Of Porcine Torque Teno Virus In Vitro And Development Of TaqMan Real-time Fluorescence Quantitative PCR For Detecting The Virus

TTV (Torque teno virus), a small, non-enveloped virus, has a single-stranded circular DNA genome and is currently classified into the family Anelloviridae. Viral DNA of both porcine TTV species (TTV1 and TTV2) has a high prevalence in both healthy ans diseased pigs worldwide and multiple infection of porcine TTV species with distinct genotypes or subtypes of the same species has been documented in China. Human TTV genome has 3900bp and porcine TTV 2700bp. So far, no porcine diseases have been found a reason-result relationship with TTV, but some porcine diseases seems have certain relationship with it such as PMWS(Post-weaning Multisystemic Wasting Syndrome) and PDNS(Porcine Dermatitis and Nephropathy Syndrome ) etc. So there are some meanings to perform researches in porcine TTV to prevent its potential pathogenicity.Since no suitable cells used in vitro culture of porcine TTV, the genome structure and mechanism of transcription and replication of porcine TTV are not clear. Three pasmids pSK+1TTV2(containing 1 copy of TTV2 genome), pSK+1.3TTV2(containing 1.3 copies of TTV2 genome) and pSK+2TTV2(containing 2 copies of TTV2 genome) were constructed and ransfected into Chang liver cells. After 48 hours, purified total RNA and digested DNA with DpnI, then amplified to be sure that no DNAs remained. After reverse-transcription, amplified cDNA with primers of ORF1, ORF2 and ORF3. By cloning and sequencing, we found that transcription product of ORF1 did not contain the complete sequence, but was spliced between 1020nt-1706nt of TTV2 genome for 687bp, which had not been predicted before; As included in ORF3, ORF2 was not determined wether trascripted although the amplified DNA band. However, we can be sure one thing that it is not spliced; ORF3 exists really and contains two discontinuous gene fragments which locate in 393nt-595nt and 1934nt-2330nt of TTV2 genome respectively, and the complete sequence of ORF3 is 600bp; ORF3 is further spliced in two forms: one takes place in 2048nt-2118nt of TTV2 genome with 71bp spliced and the coding region sequence of mRNA is 529bp, and the other appears in 2048nt-2138nt of TTV2 genome with 91bp spliced and the coding region sequence of mRNA is 509bp. So there generate two new ORFs, we call the former as ORF4 and the latter as ORF5. Analysizing further, we found that ORF3, ORF4 and ORF5 were spliced according to GT-AG rule, but ORF1 did not obey it.Two sequences in UTR of TTV1(137bp) and TTV2(139bp) which are highly conserved and highly specified are found out respectively. They are cloned to pMD18-T vector and transformated into DH5αcompetent cells, and then purified plasmids to make standards. Design primers and TaqMan probes respectively in the two sequences above. Two real-time fluorescence quantitative PCR assays for TTV1 and TTV2 respectively were established by optimizing reaction parameters and generating standard curves with a correlation coefficient of 0.984 for TTV1 and 0.994 for TTV2. The two quantitative PCR assays could both detect at least 10 copies/μL of target DNA of TTV1 or TTV2. The assays showed good specificity and no cross reaction with other porcine viruses (PRRSV, CSFV, PCV2 and SIV). The variation coefficient of intra-batch or inter-batch were both below 3%, which indicated good reproducibility. 44 clinical samples of pig were detected by the two quantitative PCR assays, and the results showed that 47.73%, 70.45% and 31.82% of the samples were positive for TTV1, TTV2, and both, respectively. Generally, the content of TTV1 DNA in samples was between 10⁵ copies/g-10⁸ copies/g and TTV2 DNA between 10⁶ copies/g-10⁷ copies/g. The established real-time fluorescence quantitative PCR assays are effective methods for investigation on epidemiology and quantitation of TTV1 and TTV2.The rich epitopes region of ORF1(ORF1a) and the whole ORF2 gene of porcine TTV2 were amplified and cloned into pCold^TMI DNA vector respectively. The two recombinant plasmids were effectively expressed in BL21(DE3)pLysS. The expressed protein of ORF1a was inclusion body and the highly pure protein were obtained by breaking with ultrasonic waves and purifying with washing buffer. For the soluble fusion protein of ORF2, the similarly highly pure protein were obtained by nickel affinity chromatograph. The polyclonal antibodies against ORF1 and ORF2 were obtained by immunizing BALB/c mice with these purified recombinant proteins describled above. And by ELISA determination their titers are both up to 1:10000. The two polyclonal antibodies are good tools for subcellular location and function of ORF1 and ORF2, construction of infectious clone and virus isolation.