Complete Genome Sequencing And Analysis Of Two Torque Teno Sus Virus

Torque teno sus virus(TTSuV)is a small circular single-stranded negative DNA virus that is widespread in pigs.There are currently two different genotypes infecting pigs,and both genotypes can be transmitted vertically and horizontally,and the virus content in the body tends to increase with age.In recent years,as researchers have continued to study the virus in depth,it has been found that the proportion of TTSuV infecting healthy animals has gradually increased.The virus itself does not cause disease immediately after infection,but the virus will reduce the ability of pigs to resist other diseases.Although the pathogenicity of the virus has not yet been determined,tests have shown that TTSuV has a promoting effect on highly pathogenic pathogens in pigs,causing significant economic losses to the breeding industry in my country.In order to understand the genetic diversity of TTSuV from pigs in Henan,this study carried out PCR detection on 11 samples of TTSuV tissue disease collected from a farm in Henan.The positive tissues were subjected to whole-genome amplification,and the whole genome of the amplified TTSuV was analyzed.Sequence genetic analysis,online software predictive analysis of the physical and chemical properties and basic structure of ORF1protein;and virus isolation and culture of positive samples.The results found that TTSuV can be successfully sub-cultured on Marc145 cells,providing data support for a better understanding of the genetic variation and growth characteristics of TTSuV,and its pathogenicity needs further research.The PCR method was used to amplify the whole genome of 10 positive samples,and the whole genome sequence of 2 strains of TTSuV1 was successfully amplified.The analysis using bioinformatics software showed that the nucleotide similarity between the two isolates was only 86.2%.The amino acid similarity is 77.7%;the nucleotide similarity between the two isolates and the TTSuV1 reference strain is between 70.3%?96.0%,and the deduced amino acid similarity is between 56.2%?90.9%,which is similar to that of the TTSuVk2reference strain.The similarity between nucleotides is less than 50%.A phylogenetic tree was constructed together with reference strains of different subtypes,and it was found that the two isolates were closely related to the TTSuV1c subtype strains and were in the same branch,that is,the two isolates were all of the TTSuV1c subtype.Analysis of gene recombination software found that both TTSuV1 isolates had gene recombination and the recombination regions were similar.The Spanish strain G21 was the parent strain,and the Chinese strain LNJZ provided the recombinant fragments.The recombination region is located in the overlapping region of ORF1 and ORF3.The He N1-A9 recombination site is located at2195-2530 bp,and the He N1-A11 recombination site is located at 2147-2650 bp.Using online software such as Net NGlyc1.0,Net OGlyc3.1 and Net Phos2.0 to predict the structure of the ORF1 protein of two isolates of TTSuV1,the results showed that the ORF1 of the two isolates consisted of 648 amino acids and did not contain O glycosylation sites The secondary structure is mainly composed of?-helix,?-sheet,random coils,etc.The He N1-A9 strain contains 4 glycosylation sites and 23 potential phosphorylation sites,while He N1-A11contains 1 N glycosylation sites and 35 potential phosphorylation sites.Virus isolation was performed on tissue disease materials that tested positive for TTSuV1,and 10 positive samples were infected with Marc145 cells for virus isolation and culture.He N1-A9 strains were blindly transmitted to the 5th generation and obvious cytopathic changes(CPE)were observed,and half of them Tissue cell infection(TCID₅₀)determination and animal pathogenicity experiments showed that the virus price of He N1-A9virus strain was about 10^5.0 TCID₅₀,and 21day piglets were challenged with 2 m L 10^5.0TCID₅₀/m L TTSuV1 virus solution.7 days after the challenge,blood was collected and PCR was performed to detect TTSuV1 positive,and the sequence was determined to be He N1-A9strain.After 28 days of rearing,no clinical symptoms were found,and no obvious lesions were found during necropsy.