Studies On Attenuation And Pathogenicity Of Swine Acute Diarrhea Syndrome Coronavirus

Swine acute diarrhea syndrome coronavirus,(SADS-Co V)is a new type of alpha coronavirus,which can cause acute diarrhea,vomiting and even death in piglets.In2017,it was the first time to identificated in pig farms in Qingyuan,Guangdong Province,China.In the early stage of SADS-Co V,the virus was isolated from the infected pig farm and purified and identified by our lab.In order to further study its biological characteristics and to find effective methods to prevent and cure the virus,the SADS-Co V-CN/GDWT/2017 strains stored in the laboratory were continuously cultured on Vero cells.At the eighth generation,it was found that the virus produced stable cytopathic effect on Vero cells and adapted to this culture mode.In the subsequent passage,plaque purification was carried out once every ten generations,and the titer of the virus was detected.The results showed that the virus price also increased with the increase of the generation.The results indicated that the cell adaptability of SADS-Co V-CN/GDWT/2017 strain was enhanced.In order to further study the reasons for the changes in biological characteristics,we cloned and analyzed the whole genome of P7,P18,P48 and P83.Sequence analysis showed that the sequence homology of the four strains was between 99.9% and 100%,and the full length of the four strains was all about 27 kb.At the same time,we found that there were 15 base mutations in the whole genome of P83 compared with P7 strain,which resulted in 9 corresponding amino acids changed.In addition,both P83 and P48 had a nucleotide deletion of 58 bp at 26619-26676 bp.Further analysis showed that the deletion of58 bp resulted in the change of 19 amino acids in the front part of the protein encoded by the NS7 a gene,and 20 additional amino acids were encoded,which might change the function of the NS7 a protein.Besides,this deletion leads to the loss of the start codon of NS7 b,which leads to the failure of NS7 b to encode the corresponding protein.Based on the above results,the pathogenicity of P83 strain was evaluated by animalexperiments.The experiment was divided into three groups: P7,P83 and control group.The results showed that there were obvious clinical symptoms in group P7 at 6 hpi,and virus shedding was detected by anal swabs on the first day after challenge;however,a piglet in group P83 only had mild diarrhea at 2 dpi.At the same time,histophagy analysis showed that the P7 piglets could detect the virus in the tonsil,stomach,duodenum,jejunum,ileum and mesenteric lymph node,among which the virus load in the ileum was the largest.P83 virus could only be detected in ileum and mesenteric lymph nodes after infection,and the virus content was very low.In order to determine that the pathological changes in the experimental group were caused by SADS-Co V,we examined the duodenum,jejunum and ileum in each group for pathological observation and immunohistochemistry.The results showed that the villi of the P7 group showed pathological breaks,shedding and shrinking,and a large number of SADS-Co V antigens were detected in the obvious area of the lesions,while the SADS-Co V antigen was almost not detected in the P83 group.There were no pathological changes in the control group.Therefore,we conclude that the virulence of P83 substrain is relatively weak,and it has little damage to animals.It was expected to be a candidate strain for preparation of attenuated vaccine.