| The Torque teno sus virus(TTSuV)is a small,non-enveloped,single-stranded,circular DNA virus that was discovered in 1997 by a Japanese hepatitis B patient after it was transplanted,and was named as TT Virus(TTV)according to the patient's name.This is the first "human circovirus" with a single-stranded circular DNA genome(ssDNA)and was classified as the family Circoviridae.Then TTV was also found in domestic animals such as pigs,chickens,cattle,sheep,cats,dogs as well as in primates such as primates and gibbons.TTSuV can be divided into two subtypes,named as TTSuV1 and TTSuV2,TTSuV1 belongs to the genus Petitavirus,and TTSuV2 belongs to the genus Kappa,both of which can infect pigs.In recent years,different countries have found TTSuV in pigs.TTSuV is mainly transmitted by animal fecal oral route,but also by vertical transmission or placental uterine transmission.TTSuV alone has no specific clinical symptoms but TTSuV is often cotransfeced with other porcine viruses such as Porcine circovirus type 2(PCV2),Porcine reproductive and respiratory syndrome virus(PRRSV),with some unknown synergistic effect,can aggravate the animal's condition.At present,TTSuV is widespread in pig farms around the world.However,the existing detection methods are limited to the viral pathogen detection by PCR method or the serological detection by ELISA method.There are no commercially available kits for the specific detection of TTSuV antibodies.Therefore,it is very important to establish a fast,efficient and sensitive antibody detection method.At the same time,there is little research on the pathogenesis of TTSuV.Therefore,to carry out sequence analysis of TTSuV virus and to establish its antibody detection method will provide a theoretical basis and technical support for the early detection and treatment of the virus.Meanwhile,it will be helpful for the typing of the mainstream strains of pigs in different regions and different pigs In-depth understanding of the virus infection in the group.1.In this study,we collected the diseased pigs from multiple swine farms around Nanyang City in Henan Province from 2016 to 2017 and genomic DNA was extracted.According to the reference sequences reported in GenBank,three pairs of primers were designed to cover the full-length of TTSuV genome 21 new TTSuV genomic sequences were obtained and named as HeN1-A1,HeN1-A2,HeN1-A6,HeN1-A7,HeN1-A8,HeN1-A9,HeN1-A10,HeN1-A11,HeN1-A12,HeN1-A31,HeN1-A32,HeN1-A51,HeN1-A52,HeN1-A132,HeN2-A2,HeN2-A4,HeN2-A5,HeN2-A9 and HeN2-A11.Sequence homology and phylogenetic tree analysis showed that the genomic sequence homology of HeN1-A2,HeN1-A6,HeN1-A8,HeN1-A9,HeN1-A11,HeN1-A31,HeN1-A52 and HeN1 was 95.7%~97.6%.The homologies of HeN1-A51,HeN1-A32,HeN1-A12,HeN1-A1 and HeN1-A10 were 84.3%~86.6 %.The homology of HeN2-A11,HeN2-A9,HeN2-A4 and HeN2-A5 was 78.8%~99%,with small difference.The homology of HeN1-A7 and HeN2-A2 were 33.2%~34.3% and 27.6%~29.6% respectively,which indicated that the homologies of these two gene sequences and other gene sequences were quite different,So separate branches in the evolutionary tree.2.In this study,we used the Cap gene sequence of TTSuV1 in GenBank to design a specific primer to amplify pcDNA3.1.The recombinant plasmid pcDNA-TTSuV1 was constructed and transfected into HEK293 T cell line.The indirect immunofluorescence of TTSuV Antibody detection method(Indirect Immunofluorescence,IFA).The results showed that the positive rates were48.8%~65.4%(65.4%,50%,48.48%,51.28% and 55.36% respectively in all the large-scale pig farms collected from 2016 to 2017 in Nanyang City,Henan Province,about50%.Indirect immunofluorescent antibody detection method of TTSuV we established has high stability and can be used for clinical testing. |
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