| Porcine Epidemic Diarrhea Virus(PEDV)is the pathogen of Porcine Epidemic Diarrhea(PED).Its clinical manifestations are watery diarrhea,vomiting and anorexia,etc.,and piglets generally die by dehydration and weight loss.PEDV can infect pigs of all ages.Pre-weaning piglets are the most infected hosts with PEDV,and their morbidity and mortality are as high as 100%.PED has seasonal epidemics,which occur frequently in winter and spring,occasionally in summer and autumn Since the outbreak of PEDV in China in 2010,its epidemic has become more widespread.The emergence of mutant strains has led to the failure of commercial vaccines to match the antigenicity of popular strains,and the immune effect is poor.It is very essential to culture PEDV in vitro for the study of PEDV.Porcine intestinal epithelial cells(IEC)are the target cells for PEDV survival in vivo and are considered to be ideal cell models for PEDV culture in vitro.As the current information on PEDV local epidemic strains is still unclear,this paper has carried out the following work to further clarify the proliferation characteristics and virulence of PEDV Anhui epidemic strains in IEC,and rabbits as experimental models,preliminary evaluate the current immune antibody levels of commercial vaccines(1)Analysis of proliferation Characteristics of PEDV AH2017 Strain in IECIn this study,PEDV was infected with IEC cells by adding was verified by RT-PCR and IFA.qPCR was used to detect the copy number of the 5th generation virusat different host post infection times,and the replication of PEDV AH2017 strain in IEC was obtained by one-step growth curve.The copy number of 5-50 generations of partial virus infected with IEC 36h was also detected by qPCR,and the change was observed by the growth curve.The strypsin,which tudy also used TEM to observe the morphology of PEDV AH2017 strain and understand its release process in IEC.The results showed that PEDV AH2017 could be stably passaged on IEC by RT-PCR,when the trypsin content was 10?g/mL during virus incubation and the trypsin content was 5 ?g/mL.The optimum concentration for PEDV AH2017 to be stably passaged on the IEC allows the PEDV AH2017 strain to be stably passed through the IEC to more than 50 generations.The one-step growth curve of the fifth generation virus of PEDV AH2017 showed that the incubation period of the virus in the target cells was about 12h,and it began to enter the rapid replication period at 24h.The IFA results showed that there was a large amount of specific fluorescence at 24 h after infection,which was further confirmed above.By analyzing the growth curve of virus 5-50 generation virus,it was found that with the increase of virus generation,the virus content was higher,indicating that the strain has strong adaptability in IEC.Electron micrographs show that PEDV is spherical and has a capsule on the outside in IEC cells.The process of germination of PEDV from cells was also observed.(2)Analysis of virulence of PEDV AH2017 strain on 2-day-old pigletsTen 2-day-old piglets without colostrum were selected for animal regression experiment.The animals were divided into experimental group and control group on average.The time of onset,and death time were counted after artificial infection of piglets.The intestinal lesions and viral colonization of piglets were observed by HE staining and immunofluorescence staining.The results indicated that the PEDV AH2017 strain infected 2-day-old piglets could cause illness within 20-24 hours,and all died within 5 days.This was not the case in the control group.It is highly pathogenic to 2-day-old piglets.HE staining showed that the villi of jejunum,ileum and duodenum of piglets were exfoliated to a certain extent,and the epithelial cells were vacuolated.It shows that PEDV AH2017 strain is highly pathogenic to 2 day old piglets.And IFA staining found that PEDV mainly colonizes epithelial cells from the jejunum and duodenum.(3)Preparation of PEDV antiserum serumIn this study,rabbits were used as animal models to initially assess immune antibody levels of current commercial vaccines and to prepare PEDV antisera.Eleven 6-week-old male rabbits were selected and divided into 4 groups,which were the immunized group and the control group.The immunization procedure applies to group A:one for attenuated vaccines and two for inactivated vaccines,3 weeks later,group B:one for inactivated vaccines and two for inactivated vaccines,3 weeks later,and C Group:one for attenuated vaccines,one for attenuated vaccines,three weeks after inactivated vaccines,three weeks after inactivated vaccines,group D:each immunized saline,the above immune channels are muscles injection.Before immunization,15 days after immunization,3d,10d,15d,20d,10d,15d,20d after the third immunization,serum was collected,and the serum antibody content of each group was detected by ELISA kit which reached a certain value and collected a large amount of blood.Separate and preserve aseptically.In group A,the antibody concentration in the serum was lower on the 20th day after the second immunization,and the immune inactivated vaccine was supplemented 3 weeks after the second immunization.The results of serum antibody test showed that the serum antibody content of group B was significantly higher than that of group A 20 days after the two immunizations.After 3 times of supplementation,the antibody content of group A was significantly higher than that of group B and C,and was significantly higher than that of group B.The above provides a preliminary understanding of the immunological effects of commercial vaccines and provides an alternative immunization procedure for the preparation of PEDV antisera.If you need antiserum in a short time,you can choose the B group immunization program;if you need a higher antibody concentration,please choose the A group immunization program.In summary,this study used PEDV natural target cell porcine small intestine villus epithelial cells(IEC)as a model,using qPCR technology to identify the growth characteristics of PEDV AH2017 strain,and the pathogenicity of the strain was analyzed by pig challenge test.force.Using rabbits as animal models,the immune antibody levels of current commercial vaccines were first evaluated and PEDV antisera were prepared.The results of this study provide a certain technical reference for the prevention and control of PED in Anhui Province.On the other hand,the study on the epidemic strains in Anhui Province provides some data support for the candidate strains for PEDV vaccine development in the future. |
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