Porcine TTV: Molecular Epidemiology, Full Length Genomic Sequence Analysis And Construction Of Infectious Molecular Clones

Torque teno virus (TTV) is small, non-enveloped virus with a single-stranded, circular DNA genome of negative polarity, belonging to the genus Anellovirus. It was first discovered in a Japanese patient with post-transfusion non-A-E hepatitis in 1997. The viruses have been detected not only in humans but in non-human vertebrates, pigs, cattles, sheep, cats, dogs, sea lions and so on.The porcine TTV which is widespread in most of swine herds in the world has attracted considerable interest. Epidemiogical study has shown that the porcine TTV partially contributed to the experimental induction of porcine dermatitis and nephropathy syndrome (PDNS) combined with porcine reproductive and respiratory syndrome virus (PRRSV) infection, and also to the experimental induction of postweaning multisystemic wasting syndrome (PMWS) combined with PCV2 infection in a gnotobiotic pig model. And TTV maybe take part in infectious hepatitis of pigs with PCV2 or hepatitis E virus (HEV). Because the porcine TTV is a potential risk to swine industry, it is necessary to study it.According to published sequences of PCV2 genomes and TTV1, 2 UTR sequences in GenBank, we designed primers and established PCR for detecting PCV2 and nested PCR for TTV1, TTV2. A total of 258 swine sera and tissues were detected. The results showed that the TTV1 and TTV2 infection rates were 37.6% and 82.6%, respectively, and their co-infection rate was 34.5%. And there are 94 samples of PCV2 and TTV1 co-infection(36.4%), there are 193 samples of PCV2 and TTV2 co-infection(74.8%), and 34.5% of the samples were identified the three viruses co-existed. We amplified UTR from some of the positive samples by nested PCR and analyzed them in comparison with the sequences of TTV1 and TTV2 previously reported. The results demonstrated that TTV could be segregated into two lineages including TTV1 and TTV2. Among our acquired sequences, UTR nucleotide sequence homologies of TTV1 or TTV2 were 80.8%-98.5% and 94.2%-100%, respectively; compared with the reference isolates, UTR homologies of TTV1 or TTV2 were 82.7%-100% and 95.5%-99.1%. In conclusion, our study provided the evidence that TTV had emerged in pigs in China, and proved that PCV2 and TTV1/2 co-infection is very common. All these indicated that TTV might be a very important new pathogen of pigs.We amplified porcine TTV1 and TTV2 full-length sequences from the samples which were TTV positive, but in sequencing we found there had been special structures and GC-rich region in porcine TTV1 and 2, making the sequencing difficult. So we designed two pairs of nested primers of porcine TTV1 and three pairs of nested primers of porcine TTV2 to amplify the full-length of porcine TTV genomic sequences. Seven full-length sequences have been successfully amplified. We compared our isolated porcine TTV sequences with the reference sequences, and the results showed that the homology between our isolated TTV1 and TTV2 was less than 10%, the homology between TTV1 isolates was 94.6-96.5%, and the homology between our TTV1 isolates and the reference sequences was 61.5-98.3%. The homology between TTV2 isolates was 92.4-94.8% and the homology between our isolates and the reference sequences was 85.4-96.2%. Amino acid sequence comparison showed that ORF1, 2, 3 amino acid homology of TTV1 isolates was 97.5%, but the homology with the reference sequences was classified into two cases, homology with AB076001 and GU456383 was 97.5%, while the homology with other four reference sequences was less than 50%. The ORF1, 2, 3 amino acid homology of TTV2 isolates was more than 88.3%, and the homology with the reference sequences were above 80%, only the homology with reference GU88046 ORF3 is exception with less than 50%. By comparison, we found that in porcine TTV, the homology of the amino acid sequences is lower than the nucleotide homology in most sequences. At the same time, in the sequences with the homology more than 90%, there exist high frequency G/A exchange. In ORF1 of porcine TTV1, the exchange rate was 40-57.6% and the exchange rate in ORF2 was 50-60%. In the ORF1 of the TTV2, there only two sequences with the homology more than 90%, the G/A exchange was 27.7% and 42.9%, in the ORF2, the exchange rate was 60%, the exchange rage of G/A were significantly higher than the others. Sequencing by using a non-methylated strain can significantly reduce the difficulties of sequencing. With this method, we obtained full-length of FJ/China/2010/TTV2/2 at once. Then we predicted DNA secondary structure and did methylation analysis. The results showed there existed complicated GC stem-loops in UTR of FJ/China/2010/TTV2/2 and but there exist no GC methylation.We expressed putative ORF1 and ORF2 in prokaryotic vector pET32a (+) and pGEX-6p-1. There exist a lot of hydrophobic amino acids in the N terminal of the ORF1, so we truncated ORF1 into ORF1a. The ORF1a protein is an inclusion body and the ORF2 protein is soluble. Through the inclusion body purification and glutathione affinity chromatography (GST) purification, we obtained high purity recombinant proteins, we immunized purified ORF1a protein and ORF2 protein into BALB/c mouse and got antiserum of 1: 6000. Then we cloned ORF1, ORF2 and ORF3 of TTV2 into eukaryotic expression vector 3×FLAG, named 3×FLAG-ORF1, ORF2 and ORF3. By transfecting them into PK15 cells, we proved our self-made antibody effective by IFA using our self-made antibody. Through the antibody of 3×FLAG, we study the protein cell localization and we observed that TTV2 ORF1 protein is localized in nuclear, ORF2 is throughout the cell, ORF3 is localized in the nuclear plasma, especially in the periphery of the nucleolus.Since the aomunt of porcine TTV in pigs is little and there are no cell lines to support the TTV replication, so the study of molecular and biological mechanisms has been limited. In order to study some of the key porcine TTV molecular and biological activity (such as transcription and replication), we amplified full length of FJ/China/2010/TTV2/2 which is 2796 nucleotides, and the homology with the earliest TTV2 isolates prototype Sd-TTV2p (AY823991) was 85.4%. And we constructed 1.0, 1.3, 2.0 full-length plasmids with pSK as the vector. By transfecting them into PK15 cells, we validated three transcriptions of porcine TTV ORFs and the occurrence of the transcription splicing of ORF3 by Northern Blot. And through the Southern Blot test based on enzyme digestion, we found there was no TTV replication in PK15 cells, and we used ORF1a and ORF2 antibody to detect the expression of the three plasmids, and the results showed that there existed ORF1 expression, but there is no signal by ORF2 antibody. This proved that the three plasmids are feasible. These experiments built a platform to study the molecular mechanisms of porcine TTV and also created conditions to find a cell line which support the TTV replication.