Studies On Molecular Biology Characters Of Porcine Torque Teno Sus Viruses And Porcine Circovirus Type2

Torque teno sus viruses (TTSuVs) and porcine circovirus type2(PCV2) are widely distributed inpigs. In recent years, some researchers found that the co-infection ratio of TTSuVs and PCV2was veryhigh. PCV2can cause porcine circovirus associated disease (PCVAD) such as postweaningmultisystemic wasting syndrome (PMWS) in pigs. However little is known about the molecular biologyof TTSuVs. In this study, we have down some research on the genetic variation, identification ofantigen epitopes, construction the infectious DNA clone and identification of promoter of TTSuV1aswell as study on the key amino acid of the conformational neutralizational epitope of PCV2capsid (Cap)protein. The purpose of this study was try to learning the the molecule biology of TTSuVs and PCV2which may be providing important information for demonstration the synergistic pathogenic of TTSuV1and PCV2in the future.Torque teno sus viruses (TTSuVs) are non-enveloped viruses and have single-stranded, negativesense circular DNA genomes. The genetic variation among different TTSuVs isolate is very large. Inorder to demonstrate the genetic variation of TTSuVs in China, in this study the TTSuV1and TTSuV2genomes were amplificated from TTSuVs positive samples by high-fidelity enzyme. The TTSuV1andTTSuV2genomes were cloned into pMD18-T vector to construction reccombiant plasmids and thensequenced. Eight TTSuV1genomes and eight TTSuV2genomes (2.9kb) were obtained in this study.The obtained TTSuVs genomes with29TTSuV1and40TTSuV2genomes in Genbank were analyzedby MEGA4.0software. Phylogenetic trees indicated that both the TTSuV1and TTSuV2sequencescould be divided into four genotypes. Within the TTSuV1a group, the similarity was between87.5%and95.2%, while in the TTSuV1b group it was83.8–99.9%and in the TTSuV1c group80.3–97.6%;but only two sequences in TTSuV1d groups which were isolated from China. The similarity of thesequences of TTSuV2a ranges between86.7%and99.7%, which is similar to TTSuV2b and TTSuV2d,while only two strains obtained in China lie within TTSuV2c. Interestingly, the sub-genotypesTTSuV1d, TTSuV2c and TTSuV2d exist only in the pig population of China. The research showed thathigh level of variation among different isolates of TTSuV1and TTSuV2existed in China.TTSuV1ORF1is considered to encode the viral Cap protein, which is crucial for the induction ofTTSuV1-specific antibodies and protective the host. Based on the results of genetic genetic variationanalysis, eight monoclonal antibody (mAbs) directed against the Cap protein of the strain in TTSuV1-csubtype were generated earlier (Liu et al.,2013). In this study, the antigen epitopes of the Cap proteinwere mapped using these mAbs and truncated Cap proteins expressed in Escherichia coli. Fine epitopemapping was then performed with a panel of synthesized polypeptides. One antigenic epitope of theCap protein, which reacted with seven mAbs, had the polypeptide sequence536HPKYAGQGGGYTT548,whereas another epitope recognized by the mAb1E9had the polypeptide sequence549EIGHQGITAASLR561. It is interesting that the two new epitopes are adjacent, but mutuallyindependent. The C-3polypeptides contain the epitope was used as antigen to detecting200pig sera samples from farms. Results showed that the antibody positive rate was81.0%. This study showed thatTTSuV1was widely spread in the pig herds of China and provided new method for further investigationof the epidemiology of TTSuV1.In order to search a cell line which TTSuV1can proliferation and rescue TTSuV1, the infectionmolecule clone of TTSuV1was constructed in this study. A special restriction enzyme site (Sal I) wasintroduced into the TTSuV1-SY genome, after digested by Sal I the TTSuV1-SY genome wasre-circored. The TTSuV1circore genomes were transfection into ST, PK-15and other cells bylipofectamineTM2000reagent according the protocol. The first generation has many TTSuV1antigenpositive cells, and from the second to the third generation, positive cells decreased, which showed theTTSuV1-SY was successfully rescued. Till the fouth generation, no positive cells were found whichshowed that ST and PK-15cells might not be fitting the culture of TTSuV1. In order to demonstrate theTTSuV1Cap protein locating in the cells, the focus microscope was used to identification. Resultsshowed that the protein of Cap was synthetized in the cytoplasm; then the Cap protein was transformedinto the nucleus. Identified by WB, the Cap protein was37kDa, which were different with thepredication Cap protein. Based on the results, we inferred that there might be existed mRNA editingphenomenon. The first generation cell culture of TTSuV1-SY was injected pigs to study the viruspathogenicity. Results showed that no significant differences were observed on temperature and weightbetween the infection group and negative control group. Only tiny lymph foilicie hyperplasia occurredin lymphoglandulae inguinales and low level cirrhosis of lung happened in the infection group. PCRdetection found that the TTSuV1DNA in lung and small intestine was more than in other tissues whichdeomonstrated that the TTSuV1could proliferation in pigs.In order to clarify the core region of TTSuV1ORF1promoter, a series primers was used toamplification the genomic fragments in leader sequence of TTSuV1ORF1. pGL3-Basic and pRL-TKvectors were used in this research. Results showed that the sequences between196nt and525nt canpromote the transcription of firefly luciferase gene which demonstrated that in the196nt and525nt hada promoter. Further research found that the core sequence of the promoter lies in250nt and400nt.After deleted this region in TTSuV1genomes, the expression of the Cap protein were affected. Thisresult provides new information on TTSuV1molecula biology.PCV2Cap protein is the main structure protein of PCV2which can induce the organism producingneutralizing antibody. A mAb8E4against the PCV2capsid protein has the capacity to neutralize thevirus. The epitope of mAb8E4was conformational epitope. However, this mAb can only react withsome PCV2a strains (LG, CL, and JF2), but does not cross-react with some PCV2b strains (YJ and JF)(Huang et al.,2011). In the present study, a mutation of arginine to alanine at position59in the capsidprotein of strain JF allowed the mutant to be recognized and neutralized by mAb8E4. Likewise,mutations of arginine to alanine at position59together with alanine to threonine at position60in thecapsid protein of the YJ strain resulted in a gain of neutralization and recognition by mAb8E4. Here,we demonstrated that the amino acids at positions59and60in the capsid protein of PCV2participate inthe formation of conformational neutralizing epitopes and mutations at positions59or59/60result in novel neutralizing epitopes of PCV2b (JF and YJ) strains. This study provides valuable information forfurther in-depth mapping of the conformational neutralizing epitopes, clarification of antigenicdifferences among PCV2strains.In summary, We clarify the genetic variation of TTSuV1in China and identified two antigenepitopes in TTSuV1Cap and established an indirect ELISA serology diagnostic method based on theepitopes; the TTSuV1infectious molecular clone was constructed and rescued the TTSuV1-SY strain;animal experiment showed that after infected pigs by the virus, there was no serious pathogenicity; apromoter of TTSuV1ORF1was identified, those provides new information for researching themolecular biology and pathogenicity of TTSuV1. Beside that, we first demonstrated that the aminoacids at positions59and60in the capsid protein of PCV2participate in the formation ofconformational neutralizing epitopes which is important for further identifying the conformationalneutralizing epitopes.