The Fill-length Genome Sequence Analysis And The Infection Model Preliminary Establishment Of The Porcine Epidemic Diarrhea Virus Strain GX15

Porcine epidemic diarrhea（PED）is a highly infectious intestinal disease caused by the Porcine epidemic diarrhea virus（PEDV）,which can quickly result in diarrhea,vomiting,dehydration,and even death.In this study,the PEDV GX15 strain isolated from Fangchenggang City,Guangxi was sequenced and the whole genome sequence was analyzed in order to understand the molecular characteristics of PEDV strains in Guangxi.Meanwhile,the cell infection model and animal infection model of the strain were established by infection tests to build a platform for PEDV pathogenesis research and the development of new vaccine and antiviral drug.Firstly,the whole genome sequence was obtained by segmented cloning.The full-length genome of the GX15 strain was 28 042 nt,belonging to the GIIb gene subtype and homologous to CH/GX/PEDV/1401/2016.Compared with the GIIb subtype reference strain AJ1102,there were 4 base mutations in the 5’UTR and 13 base insertions in the 3’UTR of the GX15strain.Except E protein and M protein,there were many variations in the deduced amino acid sequence of the coding region of GX15 strain.There were8 amino acid insertions and 56 amino acid deletions in ORF1ab.There were mutations in multiple neutralizing epitopes of S protein,and the deletion of²⁵⁹NW²⁶⁰ was also located in the CAD of the virus.There were 6 amino acid mutations in N protein.There were 2 amino acid mutations in the ORF3 protein with the molecular characteristics of PEDV highly pathogenic strain.Cell epitope prediction showed that there were four potential B-cell epitopes and eleven T cell epitopes in S protein,two B-cell epitopes and four T cell epitopes in N protein,and three T cell epitopes in M protein.These findings laid a foundation for subsequent research on the immune characteristics of GX15strain.Subsequently,the in vitro infection characteristics of GX15 strain were studied.The proliferation characteristics of the virus were analyzed using Vero cells as host cells.It was found that the optimal trypsin concentration for GX15proliferation on Vero cells was 5μg/m L,and the optimal multiplicity of infection was 0.1 MOI.The virus reached the peak of growth after 48 h of infection.The GX15 strain was further infected with RK-13,MA-104,PK-15,ST,BHK and MDBK cell lines to analyze the infectivity of GX15 strain to different cells.The cytopathic effect observation and real-time fluorescence quantitative PCR detection showed that the virus had similar proliferation characteristics with Vero cells on MA-104 cells,and the virus titer reached 10^6.12TCID₅₀/m L.The virus was also infectious to porcine cells PK15 and ST,and could effectively proliferate in these two cell lines.The virus titers were 10^4.00TCID₅₀/m L and 10^5.40 TCID₅₀/m L,showing the unique cell phagocytosis of GX15.Finally,the strain GX15 was used in the animal infection test.Six-day-old healthy piglets in different groups were infected orally with 10^4.32TCID₅₀,10^3.32TCID₅₀,10^2.32TCID₅₀,and 10^1.32TCID₅₀,respectively.The clinical symptoms changes,histopathological changes,and detoxification of piglets in different challenge groups were observed daily.The results showed that the challenge dose of 10^2.32TCID₅₀ could cause 100%animal disease.Piglets in different challenge groups began to show clinical symptoms,such as depression,weight loss,appetite loss and persistent diarrhea 1 to 2 days after inoculation.The peak of continuous detoxification through the digestive tract and respiratory tract appeared at 3～7 d and 3～5 d,respectively.Autopsy observation showed that the infected piglets had obvious gastrointestinal flatulence,thinning and swelling of the intestinal wall,and mesenteric lymphatic swelling.Histopathological observation suggested that atrophy and shedding occur in intestinal villi of the duodenum;and there were rupture in intestinal villi of the jejunum,degeneration,necrosis,and exfoliation in mucosal epithelial cell,and decrease in goblet cells.Similar pathological changes also occurred in the ileum and cecum;IHC testing showed that the GX15 strain mainly localized in the jejunum and ileum after infection in piglets.In summary,this study not only found that GX15 is a GIIb subgenotype strain of PEDV with multiple genome variations but also preliminarily established the cell infection model and animal infection model of GX15through infection test.