Genetic Variation And Biological Characteristics Of Natural Passage And Chemical Mutation Of TGEV CN12 Strain

Transmissible gastroenteritis is a highly contagious intestinal infectious disease caused by infectious gastroenteritis virus infection.The swine infectious gastroenteritis virus can cause the disease of pigs of all ages,of which the clinical symptoms of one week old piglets are the most serious and the death rate is very high.After weaning,the mortality of TGEV infected piglets was significantly lower than that of the non weaned piglets,but the infection of TGEV by weaning piglets could cause the decrease of feed reward and the situation of stiff pigs,which caused huge economic losses to the breeding industry.The mortality rate of weaned piglets infected with TGEV was lower than that of the weaned piglets.The disease was found in the United States as early as in 1946,and the disease became prevalent throughout the world.The epidemic of this disease was discovered in China in the 50s of last century.In recent years,TGEV has also been popular in China.In recent years,new TGEV strains have been found.Meanwhile,there are certain differences between the strains.In order to prevent the virus from the invasion of the pig farm,it is particularly important to study the variation of the virus and to study the vaccine of the isolated wild virus.A TGEV strain named CN 12 strain in our laboratory was successfully constructed from 120 generations to 200 generations under continuous generation,and a TGEV vaccine strain was successfully constructed.The titer of the strain was 10^7.0/mL by TCID₅₀.The continuous generation of TGEV CN12 strain was detected by indirect virus and indirect neutralization antibody test.In the experiment of indirect fluorescent antibody,the experimental group had obvious fluorescence,and there was no fluorescence in the control group.In the neutralization antibody experiment,it was found that the cells in the experimental group had no cytopathic lesions at the same time,while the control group had obvious cytopathic changes.The above experiments further confirmed that CN 12 strains were TGEV strains,and there was no presence of exogenous virus in the strain.The genomes were sequenced in the 135th generation and the 200 generation respectively.The two subbase sequences and amino acid sequences were compared with the fifth generation and 80 generation base sequences and amino acid sequences.It is found that in the long term continuous passage of TGEV CN 12,the variation of ORF1 gene and S gene is the main reason,and it is obvious that ORF1 gene is mainly replaced by base,while S gene is mainly missing or inserted.The variation of ORF1 gene was more robust,less variation and less variation in the corresponding amino acid.The variation of S gene in TGEV CN12 strain was large and its amino acid variation was large.In order to expand the selection scope of vaccine candidate strains,TGEV CN12 strain was induced by chemical mutagens methanesulfonate?EMS?from the 135 generation,and a new vaccine strain TGEV M strain was successfully obtained after mutation generation and passage to the next generation.The titer of the strain can reach 10^6.8/mL.No detection of the virus was found in the detection of exogenous virus in TGEV M strain.Sequence comparison between the 200 generation of TGEV M strain and the 135 generation of normal passage showed that ORF1 was replaced by base substitution.The deletion and insertion of S gene were the main bases,while ORF3 gene mutation existed in deletion,insertion and replacement.In the analysis,we found that the nucleotide sequence of TGEV M strain changed significantly with the increase of virus generation,which increased the base mutation of ORF3 gene,while the TGEV CN12 strain of normal passage did not change ORF3 gene.