Isolation And Identification Of Porcine Microviruses And Genome Sequence Of Tibetan Pigs In Tibet Analysis

Porcine parvovirus（PPV）can cause porcine parvovirus disease,which is a reproductive disorder characterized by infertility,abortion,stillbirth,deformed fetus,mummified fetus and weak piglets in sows,especially pregnant sows.In addition,the virus also causes diarrhea and dermatitis in newborn piglets,causing serious economic losses to the pig industry in various countries.In order to investigate the prevalence of porcine microvirus in Tibet,Tibetan pigs from four different locations in Tibet were selected as the test subjects in this experiment.Firstly,the blood samples collected from Tibetan pigs were tested by ELISA using sero-epidemiological survey,and then the PCR method for PPV detection in clinical material was established,and PPV detection was performed on the suspected material sent to the clinic,and the virus was isolated from the material with positive test results,and the biological characteristics of the isolated strains were analyzed,and on this basis,the NS1 and VP2 genes of the isolates were sequenced.The characteristics of PPV gene variation and genetic variation pattern were analyzed.The results of the study are as follows:1.ELISA was used to test a total of 356 samples of anterior vena cava blood samples collected from pigs in four different counties,different sexes,different growth stages and different health conditions in Linzhi City,Tibet,for PPV.The results showed that the positivity rates were from 100.00%in Milin County,96.55%in Bayi District,93.68%in Lang County and 72.83%in Bomi County in different areas.The positive rates at different growth stages were100.00%at the fattening stage,92.06%at the reserve stage and 83.46%at the nursery stage,in descending order.In terms of sex,the positive rate was 87.32%for boars and 96.03%for sows.Among the samples with different health conditions,100.00%were positive for diseased pigs and 90.80%for non-diseased pigs.This indicates that PPV can be detected in different environments and stages of pig growth,and the detection rate is high.2.The amplified PPV nucleic acid positive material was processed by PCR and virus isolation was performed on the lungs of Tibetan pigs to obtain a PPV isolate named XZ-PPV.The genome of XZ-PPV isolate was sequenced and a genomic sequence containing all coding regions of 4336 bp was obtained.The nucleotide sequences and amino acid sequences of the XZ-PPV isolate were compared with those of the reference strain NS1 and VP2 genes and an evolutionary tree was constructed.The results showed that the nucleotide homology of NS1 gene and the reference strain were 98.5%～99.6%,and the amino acid homology was 97.3%～99.7%.The nucleotide homology of XZ-PPV VP2 gene and the reference strain were 97.6%～99.5%,and the amino acid homology was 96%～99.5%.The genetic evolutionary tree indicated that the obtained isolates were closely related to the Chinese and German strains.Multiple sequence comparisons revealed multiple mutations in the NS1 and VP2genes,and analysis of the selection pressure on these loci revealed that several mutated loci in the VP2 gene were under positive selection pressure,which may be important for adaptation to changes in the survival environment and their own evolution.3.Bioinformatics analysis of amino acid physicochemical properties,hydrophobicity,transmembrane proteins,signal peptides and secondary structures of the isolates XZ-PPV NS1 and VP2 proteins was performed using an online website.The predicted results showed that the NS1 protein consists of662 amino acids with the molecular formula C₃₃₄₇H₅₂₂₈N₉₁₈O₁₀₁₄S₃₅,the relative molecular mass of 75673.97,the theoretical p I（isoelectric point）value of 6.64,the total number of negatively charged amino acids（Asp+Glu）of 77,the total number of positively charged amino acids（Arg+Lys）of 74,and the total The total number of atoms was 10542,the instability coefficient was 42.61,the lipid coefficient was 72.95,and the total average hydrophilic value was-0.563,making it a hydrophilic stable protein.the VP2 protein consisted of 579 amino acids with the molecular formula C₂₈₅₈H₄₃₆₁N₇₇₉O₈₈₂S₁₇,the relative molecular mass was 64290.77,and the theoretical p I（isoelectric point）value was 5.89.The total number of negatively charged amino acids（Asp+Glu）is 48,the total number of positively charged amino acids（Arg+Lys）is 38,the total atomic number is 8897,the instability coefficient is 37.16,the lipid coefficient is 67.24,and the total average hydrophilic value is-0.567,which is a hydrophilic stable protein.both NS1 protein and VP2 protein have no signal peptide and transmembrane structural domain,and in both The secondary structure of NS1protein had 294 irregular helices c,accounting for 44.41%,and the secondary structure of VP2 protein had 361 irregular helices c,accounting for 62.35%.In conclusion,this experiment used ELISA to detect porcine microviruses,and found that the detection rate of porcine microviruses in Tibet was high;one strain of porcine microvirus XZ-PPV,which was able to proliferate stably in PK-15 cells,was also isolated from the lung tissue of Tibetan pigs suspected to be infected with PPV,and the genetic evolutionary tree was constructed between the isolate and the Gen Bank reference strain,and the genetic evolutionary relationship showed that the strain was related to The genetic evolutionary relationships showed that the strain was closely related to the Chinese strain and the German strain.This study may provide a theoretical basis for the prevention and control of porcine microviruses.