Detection Method Of DHAV And Effect Of Antibody On Virus Replication

Duck virus hepatitis(DVH)is a viral congtagious disease which is of high fataliness and rapid infectivity in duckling,causing hepatitis as the main pathological characteristic and whose main pathogeny is Duck Hepatitis A Virus(DHAV)which belongs to Avihepatovirus in the family of Picornaviridae.Duck Hepatitis A Virus(DHAV)is divided into three serotypes as DHAV-A(formerly named as DHV-1),DHAV-B(isolated in Taiwan),and DHAV-C(isolated in South Korea),among which cross protection is lacked.In this study,Basing on the establishment of multiple RT-PCR and fluorescence quantitative RT-PCR detection of DHAV,the effect of specific antibody level in ducklings on the replication of artificial infection and the therapeutic effect of specific antibody at different times after virus infection were researched.It's provides guidance for prevention and treatment of duck viral hepatitis(DHAV infection).The main results of this study are as follows:1.Establishment of multiplex RT-PCR method for detect DHAV infection with different serotypesFour primers were designed and synthesized according to the whole gene sequence of duck hepatitis A virus in Genbank,and a multiplex RT-PCR method was established to detect the infection of DHAV.The method is sensitive,the minimum detection of c DNA is 10 ng.The detection of Riemerella anatipestifer(RA),Escherichia coli(E.coli)infection and RNA of healthy duckling liver tissues was negative.And no cross reactions between different serotypes.The duck with artificial infection and natural infection were in keeping with actural infection.2.Establishment of Fluorescence quantitative RT-PCR for detecting DHAVA quantitative RT-PCR method for the detection of DHAV-1 and DHAV-3 was established respectively according to the DHAV-1/3 gene sequences published in Genebank.When the method was used to detect DHAV-1,correlation coefficient of DHAV-1 was 0.998,the melting curve showed only one specific peak,and the melting temperature value is 82.5?.The sensitivity of DHAV-1 was 41 copies/?L.The correlation coefficient of DHAV-3 was 0.999,the melting curve showed only one specific peak,and the melting temperature value is 82.5?.The sensitivity of DHAV-1 was 66 copies/?L.The coincidence rate of duck with artificial infection is 100%.Among the 18 samples,8 samples were infected by DHAV-1,6 samples were infected by DHAV-3,and 4 samples were mixed infected by DHAV-1 and DHAV-3.3.The virus replication of ducklings with different levels of antibody post inoculating DHAV1-day-old ducklings were subcutaneously injected with 0.5 m L of chicken anti-DHAV-1 egg yolk antibody with a titer greater than 1:105(duck embryo neutralization titer).After 24 hours,the ducklings were given an intramuscular injection with 103 LD50(the half of the lethal dose for 1-day-old ducklings)virus.Ducklings were provided 100% protection within 75 h after infection.Fluorescence quantitative RT-PCR detection of liver tissue virus was negative,the histopathological changes of liver tissue were not obvious,and serum ALT did not change significantly.The survival rates of ducklings were 80%,60% and 10%,respectively that ducklings injected egg yolk antibody which neutralization titer were 1: 52,1: 26 and 1:17.With the decrease of antibody titer,the viral load in duck tissue was gradually increased,the pathological tissue injury was more and more serious,and the serum ALT gradually increased.The above results showed that the 0.5m L chicken anti-DHAV-1 egg yolk antibody with neutralization titer for duck embryo more than 1:105 could make 1-day-old ducklings to obtain complete protection after challenging virus.4.Evaluation of the efficacy of antibody against DHAV infection in different periodThe infection model was establishment,which 1-day-old ducklings were given an intramuscular injection in leg with 103 LD50 DHAV-1.Ducklings were subcutaneously injected with 0.5m L chicken anti-DHAV-1 egg yolk antibody with titer 1:524 at different stages of infection.The survival rate was 100%,90%,70%,30% and 20% when treatment at 5h,10 h,15h,20 h and 25 h,respectively.The viral load in duckling tissue was gradually increased,the pathological tissue injury was more and more serious,and the serum ALT gradually increased by treatment time.The above results showed that 1-day-old ducklings were fully protected by treating within 5 hours after DHAV-1 infection.