Development Of Animal Model Of PEDV Infection And Viral Load In Tested Tissues Of Piglet

Porcine Epidemic Diarrhea is a highly contagious intestinal infectious disease caused by Porcine Epklemic Diarrhea Virus,the disease mainly caused suckling piglets vomiting,diarrhea for clinical features,And the virus infected piglets can cause 100%mortality,It is one of the most common infectious diseases in the world.Since October 2010,PEDV spread spread,making the epidemic continues to expand,And the degree of harm is increasing.Vaccine immunization is an important means of PED control,but the effect is not ideal,It is sometimes reported that the immunized pig farm still breaks out the PED.We need to further scientific evaluation of PEDV vaccine immunization effect.In order to further evaluate the immune effect of PEDV vaccine,this study established PEDV infected animal model,and through the infection of piglets infected organs of the virus to identify the situation,and provide the theoretical basis for the study of PEDV infection mechanism and biosafety prevention and control.First,we established RT-PCR quantitative detection method,the primers of PEDV N suitable for RT-PCR were designed and synthesized,and construct recombinant plasmids of PEDV N,than prepared the RT-PCR standard plasmid and plotted the standard curve?Y=-3.238X+44.460:R2=0.999?.Thus,the standard copy of the PEDV virus solution was determined using the standard curve,The results showed that the virus copy concentration of the virus was 10⁸.62.62 copies/uL,This provides an accurate concentration of experimental material for the establishment of an animal model of PEDV infection.Second,this study explored the artificial feeding conditions of piglets,through the selection of different feeding tools,milk concentration and ambient temperature,the artificial feeding conditions of piglets were effectively optimized,from 60%survival rate increased to 90%.The optimization of the artificial feeding conditions effectively protects the animal model.Then,we used the virus concentration of known virus concentration of the body animal experiments.We were 10 times the gradient of the virus solution dilution(10⁰⁴),through the treatment of 20 healthy newborn non-lactating piglets,select the dilution of different dilutions of the virus on the corresponding group of experimental animals for PEDV artificial infection,by oral administration of different dilutions of the virus solution10ml to achieve the purpose of artificial infection,and then observe the clinical manifestations of experimental animals and pathological changes after dissection,to obtain the incidence of different groups of experimental animals within 48 hours after challenge,thereby calculating the EID₅₀(10^-3.5/10 ml)of the virus solution obtained,?which means that the virus suspension for 10^-3.53.5 dilution,each piglet 10ml can make 50%of the pigs infected with the disease?.This result is of great theoretical significance to the promotion of animal model.Finally,in this study,we used the constructed animal model to successfully study the infection status of different tissues and organs of 15 newborn piglets after infection with PEDV.The results showed that the infection rates of lung,spleen,liver and salivary glands were 0%,27.3%,63.6%and 63.6%respectively after PEDV infected piglets.The results of the future PED diagnosis and prevention and control and biosafety and other fields of research have important reference value.In summary,in this study,the PEDV artificial infection of healthy newborn non-lactating piglets and RT-PCR standard curve,we accurately determined the viral concentration of the EID₅₀0 of the P0EDV epidemic strain,at the same time to monitor the different organs in the PEDV infection after the drug situation.Our results are of special reference value for future important areas such as PEDV prevention and control,clinical diagnosis,vaccine research and development.